Metal-isotope-tagged monoclonal antibodies for high-dimensional mass cytometry

Abstract

Advances in single-cell mass cytometry have increasingly improved highly multidimensional characterization of immune cell heterogeneity. The immunoassay multiplexing capacity relies on monoclonal antibodies labeled with stable heavy-metal isotopes. To date, a variety of rare-earth elements and noble and post-transition metal isotopes have been used in mass cytometry; nevertheless, the methods used for antibody conjugation differ because of the individual metal coordination chemistries and distinct stabilities of various metal cations. Herein, we provide three optimized protocols for conjugating monoclonal IgG antibodies with 48 high-purity heavy-metal isotopes: (i) 38 isotopes of lanthanides, 2 isotopes of indium, and 1 isotope of yttrium; (ii) 6 isotopes of palladium; and (iii) 1 isotope of bismuth. Bifunctional chelating agents containing coordinative ligands of monomeric DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) or polymeric pentetic acid (DTPA) were used to stably sequester isotopic cations in aqueous solutions and were subsequently coupled to IgG antibodies using site-specific biorthogonal reactions. Furthermore, quantification methods based on antibody inherent absorption at 280 nm and on extrinsic absorption at 562 nm after staining with bicinchoninic acid (BCA) are reported to determine metal-isotope-tagged antibodies. In addition, a freeze-drying procedure to prepare palladium isotopic mass tags is described. To demonstrate the utility, experiments using six palladium-tagged CD45 antibodies for barcoding assays of live immune cells in cytometry by time-of-flight (CyTOF) are described. Conjugation of pure isotopes of lanthanides, indium, or yttrium takes ~3.5 h. Conjugation of bismuth takes ~4 h. Preparation of palladium mass tags takes ~8 h. Conjugation of pure isotopes of palladium takes ~2.5 h. Antibody titration takes ~4 h.

Introduction

Elemental mass tags in CyTOF

Cytometric technology has facilitated the investigation of multiple features of heterogeneous biological systems at the single-cell level^1,2. Mass cytometry, or CyTOF, has been regarded as the ‘next generation’ of flow cytometry because it uses metal-isotope-tagged antibodies (MitAbs) as reporting probes^3–6. Using this advance, CyTOF overcomes the inherent limits of spectral overlap observed with conventional fluorescence-based cytometry and enables simultaneous measurements of >50 parameters in single cells^7–12. In principle, mass cytometry originates from inductively coupled plasma TOF mass spectrometry (ICP-TOF-MS), which is broadly used for quantifying isotopic contents of elements having low ionization potentials in the periodic table^13–15. To enable biomedical assays, elemental mass tags (EMTs) have been introduced for CyTOF to label biomolecular targets or probes of interest with specific elements or isotopes. A quantitative relationship is established between the concentration of targeted biomolecules and the intensity of the detection signal of the corresponding EMTs^16–22.

To minimize background signals, EMTs are generally composed of exogenous cellular elements such as noble and post-transition metals, rare-earth elements, and halogens, rather than the essential elements of endogenous cellular components such as sodium, potassium, calcium, copper, iron, and zinc. To date, the tagging elements and isotopes investigated in CyTOF have involved yttrium (Y)^23,24, indium (In)^4,25, the series of lanthanide elements (Ln, from La to Lu, except Pm)^4,9,26, iodine (I)²⁷, cadmium (Cd)^4,21, tellurium (Te)^28–30, silver (Ag)³¹, palladium (Pd)^25,32,33, rhodium (Rh)³⁴, iridium (Ir)³⁴, platinum (Pt)^35,36, ruthenium (Ru)^37,38, osmium (Os)³⁸, and bismuth (Bi)^39,40. Basically, EMTs have two fundamental utilities: (i) measurement of the expressions of cellular proteins or their modifications and (ii) characterization of cell functions relating to viability³⁵ and cell cycle²⁷, as well as for high-throughput cell barcode³². Figure 1 illustrates the most widely used and commercially available EMTs in current CyTOF applications. The different utilities of EMTs are dependent on their chelation properties in antibody conjugation or biochemical functions in cell biology, such as incorporation of 5-iodo-2′-deoxyuridine (IdU) into the newly synthesized nucleic acids²⁷. The metal isotopes used in protein-detection mass tags require substantial validation, as the presence of these tagged metallic cations may alter the specificity of antibody to recognize its antigen⁴¹.

Fig. 1 |. Elemental mass tags widely applied in CyTOF.

Elemental mass tags are typically classified as protein-detection mass tags or cell-identification mass tags according to their utilities in CyTOF analysis. To date, 56 stable heavy isotopes have been used in single-cell assays, coming from 15 rare-earth elements (Y, and from La to Lu, except Pm), four noble metals (Rh, Pd, Ir, and Pt), two post-transition metals (In and Bi), and one halogen (I).

Metal isotopes used in protein-detection mass tags

Although CyTOF has minimal overlap signals compared with fluorescence-based cytometry, the spillover effects still exist among different detection channels⁴². Therefore, the preparation of MitAbs must be evaluated for (i) impurities in the enriched isotopes; (ii) ‘M ± 1’ spillover between neighbor channels, referred as abundance sensitivity; and (iii) ‘M + 16’ spillover of oxide formation. To date, 48 isotopes coming from 18 heavy metals have been conjugated to monoclonal IgG antibodies and are widely used in single-cell CyTOF immunoassays. The isotopic purities of Y, Pd, In, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, and Bi, which are typically used in our lab (https://nolanlab.stanford.edu/cytofisotopes), are shown in Fig. 2. The purified isotopes are produced using electromagnetic separation by Trace Science International. Most of these isotopes are enriched by >95% and some are available at purities higher than 99%. There are six monoisotopic elements that show 100% abundance: ⁸⁹Y, ¹⁴¹Pr, ¹⁵⁹Tb, ¹⁶⁵Ho, ¹⁶⁹Tm, and ²⁰⁹Bi.

Fig. 2 |. Pure metal isotopes tagged in monoclonal antibodies.

Forty-eight stable metal isotopes of Y, Pd, In, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, and Bi have been used in protein-detection mass tags. The typical values of isotopic enrichment and spillover impurity are based on the product sheets from Trace Science International and early ICP-MS analytical measurements performed by DVS Sciences and our lab.

The series of lanthanides are the most widely used for EMTs because they have similar chemical properties and 38 stable isotopes that can be conjugated to antibodies using the same protocol. An issue with lanthanides is that oxide formation of the light lanthanides (from 139 to 160 amu) causes mass interferences with the heavy lanthanides (from 155 to 176 amu). This is referred to as the ‘M + 16’ effect. For example, ¹⁴¹Pr¹⁶O⁺ ions have spillover signal in the ¹⁵⁷Gd⁺ detection channel. Table 1 shows the oxide species of light lanthanides and the relative ratios⁴³ that result in spillover signals in the CyTOF detection channels of the heavy lanthanides. In CyTOF tuning procedures, radiofrequency power, flow speeds and makeup of nebulization gas, and positions of the sample torch and cone can be optimized so that the oxidation ratio of lanthanum (¹³⁹La¹⁶O⁺/¹³⁹La⁺), the most easily oxidized isotope, remains <3.0%^3,18. Isotopes of ⁸⁹Y, ¹⁰²Pd, ¹⁰⁴Pd, ¹⁰⁵Pd, ¹⁰⁶Pd, ¹⁰⁸Pd, ¹¹⁰Pd, ¹¹³In, ¹¹⁵In, and ²⁰⁹Bi do not generate oxidation mass interferences with each other or with light or heavy lanthanides.

Table 1 |.

The oxides and their ratios of light lanthanides, affected CyTOF detection channels, and overlapped heavy lanthanide ions

Light lanthanides			Heavy lanthanides
Interfering elements	One positive charge oxide ions	Ratios of oxides (%)a	Mass spectrum overlapped ions	Detection channels (m/z)
Lanthanum	139La16O+	≤3.0	155Gd+	155
Cerium	140Ce16O+	≤3.0	156Gd+	156
Praseodymium	141Pr16O+	≤2.4	157Gd+	157
Neodymium	142Nd16O+	≤2.1	158Gd+	158
	143Nd16O+		159Tb+	159
	144Nd16O+		160Gd+	160
	145Nd16O+		161Dy+	161
	146Nd16O+		162Dy+	162
	148Nd16O+		164Dy+	164
	150Nd16O+		166Er+	166
Samarium	147Sm16O+	≤0.5	163Dy+	163
	149Sm16O+		165Ho+	165
	152Sm16O+		168Er+	168
	154Sm16O+		170Er+	170
Europium	151Eu16O+	≤0.1	167Er+	167
	153Eu16O+		169Tm+	169
Gadolinium	155Gd16O+	≤1.5	171Yb+	171
	156Gd16O+		172Yb+	172
	157Gd16O+		173Yb+	173
	158Gd16O+		174Yb+	174
	160Gd16O+		176Yb+	176
Terbium	159Tb16O+	≤1.2	175Lu+	175

^aThe values are based on the published ratios of lanthanide oxides measured by inductively coupled plasma mass spectrometry analysis⁴³.

Comparison of metal isotope chelation methods

In order to site-specifically attach metal cations to an antibody, bifunctional chelating agents (BFCAs, Fig. 3a,b) are used. These agents consist of two functional groups: (i) the coordinative chelator, either DOTA or DTPA, which enables chelation of metal cations; and (ii) a maleimide-functionalized group, which enables coupling to sulfhydryl groups located in the hinge region between two heavy chains of the IgG antibody that is partially reduced under mild conditions. As some monoclonal antibodies have primary amine groups within the antigen-binding sites, sulfhydryl-reactive BFCAs have less effect on antigen-binding affinities of antibodies than do amine-reactive BFCAs. To increase the number of metal-chelating cations, polymeric BFCAs with multiple chelation sites along the backbone are developed^44–46.

Fig. 3 |. Bifunctional chelating agents and corresponding conjugated antibodies.

a,b, Two types of bifunctional chelating agents (BFCAs) are complexed with tagging metal cations: monomeric DOTA (a) and polymeric DTPA (b). The selected antibody (Ab) is coupled with BFCAs via a biorthogonal reaction of maleimide moieties and sulfhydryl groups of cysteine residues in the hinge region between two heavy chains. c,d, Antibody conjugated with monomeric BFCAs (mBFCAs) (c) and antibody conjugated with polymeric BFCAs (pBFCAs) (d).

DOTA and DTPA are polyaminocarboxlyates with multiple oxygen and nitrogen donors that can form stable complexes with trivalent cations of Y, In, Ln, and Bi. The stability constant values (log K) of the complexes of DOTA and lanthanides in water are >22.8 (ref.⁴⁷). It is important that the buffers used for chelating these cations be substantially different. Y³⁺, Ln³⁺, and Ln³⁺ cations are stable in ammonium acetate buffer (pH 5.5 to 6.0), whereas Bi³⁺ cations will precipitate in this buffer because of hydrolysis. The optimal condition for chelating Bi³⁺ cation to DTPA is placement in 2–5% (vol/vol) nitric acid. The complex of DTPA with Bi³⁺ cation has a distinct feature of the UV-visible absorption spectrum in the range from 250 to 300 nm, which interferes with protein absorption at 280 nm. As a result, although the same BFCA (Maxpar X8 polymer) is used, the antibody conjugation protocol and quantification method for Bi are different from those of Y, In, and Ln, as shown in Fig. 3d.

Polymeric BFCAs can label more metal cations to an antibody than monomeric BFCAs can; however, macromolecular structure and large stereoscopic volumes may affect antibody recognition ability of the antigen. It has been reported that antibodies conjugated with Pd²⁺ cations using polymeric BFCAs lose their immune specificities⁴¹. Conversely, the loss of antigen-binding specificity does not appear to be an issue when using monomeric BFCAs that have smaller molecular structures, e.g., isothiocyanobenzyl-EDTA (SCN-Bn-EDTA, molecular weight (MW) 439 Da) was successfully used to conjugate CD45 antibodies with palladium isotopic cations for barcoding assays of live cells²⁵. A previous study of palladium complexes of aminopolycarboxylic ligands in aqueous solution reported that DTPA complex with Pd²⁺ is more stable (log K of 36.31) than EDTA complex with Pd²⁺ (log K of 23.60)⁴⁸. Therefore, in this presented protocol, monomeric BFCA of maleimide-functionalized-DOTA was used to chelate Pd²⁺ isotopic cations and was conjugated to the monoclonal antibody, as illustrated in Fig. 3a,c.

Limitations of metal-isotope-tagged antibodies

The newest version of CyTOF (Helios) can simultaneously detect 135 channels with a mass range from 75 to 209 Da. However, the number of channels used to measure surface or intercellular proteins is currently <50, because of the lack of the available mass tags. Although nanoparticle-based mass tags such as metal-embedding nanocrystals, quantum dots, and polymer dots have been considered promising^49–53, their routine usage in mass cytometry for single-cell immunoassays still requires more validation because of the inherent issue of nanoparticles, i.e., nonspecific physical absorption easily produces false-positive signals in negative-control cells. Furthermore, the large stereoscopic volumes of nanoparticles will result in inefficient diffusion through cell membranes.

At present, detection sensitivities in most of MitAbs are comparable to those of fluorochrome-tagged antibodies but are lower than those using high-sensitive fluorescent reporters such as phyco-erythrin (PE) dyes¹³. It is necessary to increase the number of tagged metal cations per antibody for a lower limit of detection. In addition, it should be noted that the protocols presented here are suitable only for IgG immunoglobulins. The protocols involve the reduction procedure of disulfide bonds, which may alter the abilities of IgE or IgM immunoglobulins that contain cross-linking disulfide bonds for their immunity structures.

Applications in single-cell CyTOF immunoassays

In mass cytometry, MitAbs have been widely used to detect cell surface, cytoplasmic, and nuclear proteins for immune phenotyping and signaling pathway profiling. The single-cell CyTOF-based investigations have been involved in the fields of basic and clinic immunology^54–59, cancer cell biology^60–63, stem cell biology^64–66, pharmaceutical development^67–69, and elemental biodistribution research^70–73. The detection sensitivity of a particular MitAb is dependent on the ionization potential and the ion transmission efficiency of the tagged metal isotope. To obtain high signal-to-noise ratios, antibody panels are optimized to pair antibodies against different expression-level biomarkers with metal isotopes of different sensitivities^23,74,75. Two general guidelines are followed. First, biomarkers expected to be of high abundance and thus readily detected (e.g., CD45, CD3, and CD20) are paired with low-sensitivity isotopes (⁸⁹Y, ^102–110Pd, ¹¹³In, and ¹¹⁵In) or medium-sensitivity isotopes (^139–152Ln). Those biomarkers expected to be of low abundance or difficult to detect (e.g., CD56, p-STAT3, and interferon-γ (IFNγ)) are paired with high-sensitivity isotopes (^153–176Ln and ²⁰⁹Bi). Second, care should be taken to avoid pairing a low-abundance biomarker with an isotope that has overlap with the oxide of an isotope conjugated to a high-abundance biomarker.

Experimental design

We describe three procedures for conjugation of metal mass tags to monoclonal antibodies for use in high-dimensional mass cytometry. These provide detailed descriptions of the preparation of metal isotopic mass tags, conjugation of IgG antibodies with 48 pure metal isotopes, and quantification methods for the conjugated antibodies. Equipment, including a lyophilizer and mass cytometers, is shown in Fig. 4. Procedures for single-cell immunoassays of CyTOF are also described.

Fig. 4 |. Materials and equipment.

a, Liquid nitrogen tank for snap freezing of the solutions of Pd@mal-DOTA complexes, PCR tubes in a 96-well metal block, dry ice for placing frozen sample PCR tubes, and Drierite desiccant for storing dried palladium mass tags to protect the from moisture. b, Lyophilizer and pump for ice-drying of palladium mass tags. c, CyTOF instruments of v1, v2, and v3 (Helios) used in our lab.

Antibody conjugation and quantification

Box 1 describes the preparation of monomeric BFCA mass tags containing six palladium isotopes using a freeze-drying process. The polymeric BFCA Maxpar X8 polymers were used to conjugate antibodies with the other 42 metal isotopes. Box 2 describes two quantification methods to determine the concentrations of conjugated antibodies. Sections 1, 2, and 3 in the Procedure describe the conjugation of IgG antibodies with metal isotopes of (i) palladium; (ii) lanthanide, yttrium, and indium; and (iii) bismuth, respectively. The timings for these conjugations are summarized in Fig. 5.

Box 1 |. Preparation of palladium isotopic mass tags ● Timing 8 h.

For the conjugation of monoclonal antibodies with six palladium isotopes, as described in Section 1 of the Procedure, mass tags of Pd@mal-DOTA are prepared using the materials and equipment shown in Fig. 4a,b.

Additional reagents

• Isotope-enriched palladium nitrates with the following purities: ¹⁰²Pd (91%), ¹⁰⁴Pd (96%), ¹⁰⁵Pd (98%), ¹⁰⁶Pd (99%), ¹⁰⁸Pd (99%), and ¹¹⁰Pd (99%) (Trace Sciences International)

  ! CAUTION Palladium nitrate is toxic. Avoid contact with skin and eyes.

• Maleimido-mono-amide-DOTA (mal-DOTA; Macrocyclics, cat. no. B-272)

• Hydrochloric acid (36.5–38.0% (wt/wt) HCl; Sigma, cat. no. H1758–100ML)

  ! CAUTION Hydrochloric acid is corrosive. Avoid contact with skin and eyes.

• Liquid nitrogen

  ! CAUTION Liquid nitrogen has a boiling temperature of −196 °C at atmospheric pressure. Direct contact can freeze the skin, causing frostbite and cold burns. Protect the hands at all times with cryogenic gloves when handling with liquid nitrogen.

• Dry ice

• Drierite desiccant (VWR, cat. no. 22891–050)

Additional instruments

• Lyophilizer (Labconco, model no. FreeZone 4.5)

• Electrospray ionization mass spectrometer (Waters, model no. Micromass ZQ)

Procedure

1. Dissolve the isotope-enriched palladium nitrates to a 50 mM concentration in hydrochloric acid. Solutions will be dark brown and transparent. Store the solutions for up to 2 years at 4 °C.

2. Weigh out ~25 mg of solid mal-DOTA powder into a 2.0-mL Eppendorf tube. Add an appropriate volume of double-distilled water (ddH₂O), then mix well to obtain a 25 mM mal-DOTA solution.

   ▲ ACRITICAL STEP The aqueous mal-DOTA solution is unstable. The solution should be prepared immediately before use.

3. Pipette 24 μL of 25 mM mal-DOTA solution and 10 μL of 50 mM palladium solution into a 0.2-mL PCR tube. The molar ratio of DOTA to Pd is 1.2:1. The slight excess of DOTA chelator is used to avoid free palladium cations in the solution.

4. Cap the tube and vortex to mix. Spin at 1,000g for 10 s at room temperature (RT, ~25°C) in a tabletop microcentrifuge.

5. Immediately snap-freeze the mixture of mal-DOTA and palladium in liquid nitrogen. Keep the PCR tube upright to ensure that the mixture remains at the bottom of the tube.

6. Place the frozen PCR tubes into a pre-cooled 96-well metal block sitting on dry ice.

7. Repeat steps 3–6 for each isotope of palladium to be used. All isotopes should be prepared as quickly as possible.

8. Replace the original caps of all the PCR tubes with caps pierced with a wide-gauge needle.

9. Lyophilize the mixtures for at least 6 h. This will yield brown or orange solids.

10. Replace the vented caps with intact caps and place the PCR tubes in a Ziploc bag containing 25 g of Drierite desiccant to protect from moisture.

    ■ PAUSE POINT The lyophilized mass tags can be stored for up to 2 years at −80 °C.

11. Dissolve the lyophilized mixtures in 50 μL of dimethyl sulfoxide (DMSO), yielding 10 mM solutions of Pd@mal-DOTA mass tags.

12. Divide the mass tags into aliquots of appropriate volume.

    ■ PAUSE POINT Palladium isotopic mass tags can be stored for up to 1 year at −20 °C.

    ? TROUBLESHOOTING

13. Analyze the palladium isotopic mass tags using ESI-MS in negative-ion mode.

    ▲ ACRITICAL STEP Because mal-DOTA is a triprotic acid and Pd@mal-DOTA is a monoprotic acid, the negative-ion ESI-MS mode should be used for all measurements.

Box 2 |. Quantification of metal-isotope-tagged antibodies ● Timing 1 h.

Two methods are described to quantify MitAbs: an assay based on absorbance at 280 nm (A₂₈₀) (method A) and a bicinchoninic acid (BCA)-based assay (method B). The A₂₈₀-based method does not require the generation of a standard curve. The molar extinction coefficient of a typical mammalian IgG antibody with a MW of 150,000 Da is 210,000 M/cm, which is equal to a percentage extinction coefficient of 13.7 for a 1% (wt/vol) (i.e., 10 mg/mL) IgG solution. This assay can be used to quantify the antibodies conjugated with the isotopes of lanthanides, indium, yttrium, and palladium, but not bismuth. The BCA-based method used for the quantification of Bi-tagged antibody requires staining with BCA dyes and measurement of UV-visible absorbance at 562 nm (A₅₆₂). A calibration curve of A₅₆₂ versus antibody concentration must be generated using IgG standards of known concentrations.

Additional reagents

• BCA protein assay reagent A (Thermo Fisher Scientific, cat. no. 23225)

• BCA protein assay reagent B (Thermo Fisher Scientific, cat. no. 23225)

• IgG control antibody (1.0 mg; R&D Systems, cat. no. MAB004)

Reagent setup

• BCA working solution. Combine 1,000 μL of BCA protein assay reagent A with 20 μL of BCA protein assay reagent B (50:1, reagent A/B), then mix well by vortexing. The solution should be freshly prepared.

• IgG antibody standards. Dissolve 1.0 mg of lyophilized IgG control antibody in 1.0 mL of phosphate-buffered saline (PBS) to obtain a 1,000 μg/mL IgG antibody standard. Prepare a series of diluted IgG antibody standards at concentrations of 100, 200, 500, and 750 μg/mL. Store at 4 °C for up to 1 year.

Additional instruments

• UV-visible spectrophotometer (Thermo Fisher Scientific, model no. NanoDrop 2000)

Method A (A₂₈₀-based quantitative assay)

1. Zero the NanoDrop spectrophotometer with W-buffer.

2. Pipette 2.0 μL of Y-, Pd-, In-, or Ln-tagged antibody onto the measurement pedestal.

3. Measure the absorbance of each conjugated antibody at 280 nm (A₂₈₀).

4. Calculate the concentration of the conjugated antibody using the average A₂₈₀ value of three measurements. A concentration of 1,000 μg/mL corresponds to an A₂₈₀ value of 1.37 absorbance units.

Method B (BCA-based quantitative assay)

1. Pipette 2.0 μL of each IgG antibody standard and Bi-tagged antibody into separate pre-labeled PCR tubes.

2. Add 30 μL of pre-prepared BCA working solution to each PCR tube and mix well by pipetting up and down slowly to avoid microbubbles.

3. Incubate the mixtures at 37 °C in a water bath for 30 min.

4. Cool all PCR tubes to RT.

5. Zero the NanoDrop spectrophotometer with ddH₂O.

6. Measure the absorbance at 562 nm (A₅₆₂) of all standards and Bi-tagged antibody within 10 min.

   ▲ CRITICAL STEP As the BCA assay does not reach a true end point, color development will slowly continue at RT. If all measurements of A₅₆₂ are completed within 10 min, no substantial error will be introduced.

7. Plot the intensity of the A₅₆₂ value of each IgG antibody standard versus its concentration to generate a calibration curve for the quantitative analysis.

8. Calculate the concentration of Bi-tagged antibody using the average A₅₆₂ value from three measurements.

   ? TROUBLESHOOTING

Fig. 5 |. Timing for conjugating antibodies with different metal isotopes.

a, Steps for conjugation of palladium isotopic mass tags. b, Steps for conjugation of yttrium, indium, or lanthanide isotopic mass tags. c, Steps for conjugation of bismuth mass tag.

Characterization and applications of palladium isotopic mass tags

To characterize the synthesized palladium isotopic mass tags (Pd@mal-DOTA), consisting of palladium isotopic cations (Pd²⁺) and maleimido-mono-amide-DOTA (mal-DOTA), single quadrupole electrospray ionization mass spectrometry (ESI-MS) was used to identify the MW of each palladium isotopic complex, as shown in Fig. 6. Because palladium isotopes have low sensitivities in CyTOF and monomeric mal-DOTA is used to conjugate antibody, palladium isotopic mass tags are suitable for pairing with cellular biomarkers expressed at high levels, such as CD45 in immune cells. In mass cytometry, live-cell barcoding is very important for a reliable comparison between experimental samples; this high-throughput technology overcomes variations in cell staining and CyTOF measurements. Therefore, to demonstrate the utility of palladium isotopic mass tags, CD45 antibody was conjugated with ¹⁰²Pd, ¹⁰⁴Pd, ¹⁰⁵Pd, ¹⁰⁶Pd, ¹⁰⁸Pd, and ¹¹⁰Pd, and was used for live Jurkat cell barcoding. In addition, three strategies for staining combinations were designed for multiplexed barcoding: (i) 6-choose-1, with 6 multiplexing; (ii) 6-choose-2, with 15 multiplexing; and (iii) 6-choose-3, with 20 multiplexing. The measurements of CyTOF ‘rain dots’ and analysis of biaxial contour plots are shown in Fig. 7.

Fig. 6 |. Mass spectra of palladium isotopic mass tags.

a–g, Negative-ion mode mass spectra of mal-DOTA, expected peak m/z = 525.2 (a), ¹⁰²Pd@mal-DOTA, expected peak m/z = 625.2 (b), ¹⁰⁴Pd@mal-DOTA, expected peak m/z = 627.2 (c), ¹⁰⁵Pd@mal-DOTA, expected peak m/z = 628.2 (d), ¹⁰⁶Pd@mal-DOTA, expected peak m/z = 629.2 (e), ¹⁰⁸Pd@mal-DOTA, expected peak m/z = 631.2 (f), and ¹¹⁰Pd@mal-DOTA, expected peak m/z = 633.2 (g). MW, molecular weight.

Fig. 7 |. Barcoding live Jurkat cells using Pd-tagged CD45 antibodies.

Live Jurkat cells were barcoded using three strategies for combining CD45 antibodies tagged with six palladium isotopes. The top section of each panel shows the CyTOF measurements as ‘rain dots’. The bottom sections show the biaxial contour plots of the indicated isotopes. a, 6-choose-1 barcoding strategy: Jurkat cells were stained with each of the six Pd-tagged CD45 antibodies. b, 6-choose-2 barcoding strategy: Jurkat cells were stained with ¹⁰⁶Pd- and ¹⁰⁸Pd-tagged CD45 antibodies, ¹⁰⁸Pd- and ¹¹⁰Pd-tagged CD45 antibodies, or ¹⁰⁶Pd- and ¹¹⁰Pd-tagged CD45 antibodies. c, 6-choose-3 barcoding strategy: Jurkat cells were stained with ¹⁰²Pd-, ¹⁰⁴Pd-, and ¹⁰⁵Pd-tagged CD45 antibodies.

Titrations of surface and intracellular antibodies

It is critical to choose appropriate cell populations as positive and negative controls to validate and titrate the conjugated antibodies. When using samples of human or mouse peripheral blood mononuclear cells (PBMCs), the positive and negative controls can be identified by gating the subset populations, e.g., to validate an antibody against human CD3, the positive control could be CD2⁺CD5⁺ human T cells and the negative control could be CD19⁺CD20⁺ human B cells, or to validate an antibody against mouse MHC class II, the positive control could be CD4⁻CD8⁻CD45R⁺ mouse B cells and the negative control could be CD4⁺or CD8⁺ mouse T cells. For antibodies against stimulation-inducible intracellular molecules (e.g., pSTAT3) or against secreted molecules (e.g., IFN-γ), appropriate stimulation conditions (e.g., interleukin (IL)-10, IL-12, or IL-18) are applied as positive controls, as shown in Fig. 8.

Fig. 8 |. Titrations of various ²⁰⁹Bi-tagged antibodies.

a, Titration histograms of ²⁰⁹Bi-tagged MHC class II antibody with mouse PBMCs and titration curves in B cells and T cells. b–e, Titration histograms at the optimal concentrations. b, ²⁰⁹Bi-tagged p-STAT3 antibody of 1,000 ng/mL with CD33⁺CD14⁺ monocytes in human PBMCs with stimulation of 100 ng/mL IL-10 as positive control and without stimulation as negative control. c, ²⁰⁹Bi-tagged p-histone H3 antibody of 2,000 ng/mL with mesodermal progenitors derived from human embryonic stem cells on day 6 of differentiation as positive control and cardiomyocytes derived from human embryonic stem cells as negative control. d, ²⁰⁹Bi-tagged IFN-γ antibody of 1,000 ng/mL with natural killer cells with stimulation of 50 ng/mL IL-12 and IL-18 as positive control and without stimulation as negative control. e, ²⁰⁹Bi-tagged anti-PE antibody of 1,000 ng/mL with PE-conjugated CD3-antibody-stained T cells and B cells in human PBMCs as positive and negative controls, respectively. cps, counts per second.

Comparisons of bismuth- and lanthanide-tagged antibodies

To validate the measurement accuracy of bismuth-tagged antibody in high-dimensional CyTOF assays, the same antibodies were conjugated with both bismuth and lanthanide mass tags: (i) human anti-CD3 tagged with ²⁰⁹Bi and ¹⁷⁰Er, (ii) human anti-CD56 tagged with ²⁰⁹Bi and ¹⁷⁶Yb, and (iii) mouse anti-MHC class II tagged with ²⁰⁹Bi and ¹⁷⁶Yb. Experiments were carried out in Jurkat cells, human PBMCs, and immune cells of mouse organs. The comparisons of the analysis results using biaxial scatter plots, heat maps, viSNE plots, and scaffold maps were performed as shown in Figs. 9 and 10.

Fig. 9 |. Comparisons of bismuth- and lanthanide-tagged antibodies in single-cell assays.

a, Left, Comparable biaxial scatter plots of CD3 versus CD19, CD4, CD11a, CD5, CD7, CD184, and CD2 in Jurkat cells stained with either ²⁰⁹Bi- or ¹⁷⁰Er-tagged CD3. a, Right, Data analysis from plots at left, depicted in radar charts of the mean intensities of the seven markers. b, Comparable immunophenotypic gating as shown by viSNE plots of data from human PMBCs stained for 40 markers and either ²⁰⁹Bi- or ¹⁷⁶Yb-tagged CD56. Fifteen cell subsets were identified (gating hierarchy described in Supplementary Fig. 2). c, Data from b depicted with heat maps. There is a strong correlation between data collected using ²⁰⁹Bi- and ¹⁷⁶Yb-tagged CD56 (Pearson correlation, r = 0.991, P < 0.00001, Supplementary Fig. 1b).

Fig. 10 |. Application of ²⁰⁹Bi-tagged MHC class II antibody in systematic immune analysis.

a, Measurements of expression of MHC class II in different mouse organ cells. b, Comparison of ²⁰⁹Bi- and ¹⁷⁶Yb-tagged MHC class II antibodies in scaffold maps, revealing immune organization of the bone marrow for C57BL/6 mice. Red nodes denote landmark manually gated cell populations; blue nodes present unsupervised cell clusters from the same data. Data in left panel of b from ref.⁹. gd T cells, γδ T cells; HSC, hematopoietic stem cell; mDCs, myeloid dendritic cells; NK, natural killer; NKT, natural killer T; pDCs, plasmacytoid dendritic cells.

Materials

Reagents

Palladium conjugation

• Tris-(2-carboxyethyl)phosphine hydrochloride (TCEP; Thermo Fisher Scientific, cat. no. 77720)

• C-buffer for antibody conjugation (Maxpar Labeling Kits; Fluidigm)

• W-buffer for washing conjugated antibodies (Maxpar Labeling Kits; Fluidigm)

• Antibody stabilizer (Candor Biosciences, cat. no. 131050)

• Zeba 7-kDa 0.5-mL spin desalting columns (Thermo Fisher Scientific, cat. no. 89883)

Lanthanide, yttrium, and indium conjugation

• Tris-(2-carboxyethyl)phosphine hydrochloride (TCEP; Thermo Fisher Scientific, cat. no. 77720)

• Maxpar X8 polymer (Maxpar Labeling Kits; Fluidigm)

• L-buffer for chelation of polymers (Maxpar Labeling Kits; Fluidigm)

• R-buffer for partial reduction of antibodies (Maxpar Labeling Kits; Fluidigm)

• C-buffer for antibody conjugation (Maxpar Labeling Kits; Fluidigm)

• W-buffer for washing conjugated antibodies (Maxpar Labeling Kits; Fluidigm)

• Antibody stabilizer (Candor Biosciences, cat. no. 131050)

• Isotope-enriched lanthanide (III) solution (Fluidigm, catalog number varies by isotope)

• Isotope-enriched lanthanide (III) nitrates or chlorides (Trace Sciences International)

• Yttrium (III) nitrate tetrahydrate (Sigma-Aldrich, cat. no. 217239–10G)

• Isotope-enriched indium (III) chlorides (Trace Sciences International)

Bismuth conjugation

• Tris-(2-carboxyethyl)phosphine hydrochloride (TCEP; Thermo Fisher Scientific, cat. no. 77720)

• Maxpar X8 polymer (Maxpar Labeling Kits; Fluidigm)

• R-buffer for partial reduction of antibodies (Maxpar Labeling Kits; Fluidigm)

• C-buffer for antibody conjugation (Maxpar Labeling Kits; Fluidigm)

• W-buffer for washing conjugated antibodies (Maxpar Labeling Kits; Fluidigm)

• Antibody stabilizer (Candor Biosciences, cat. no. 131050)

• Nitric acid (HNO₃; Sigma-Aldrich, cat. no. 225711)

• Bismuth (III) nitrate pentahydrate (Sigma-Aldrich, cat. no. 254150)

Purified antibodies

• 100 μg of IgG antibody in low-sodium azide buffer (carrier-free) with specificity of choice, such as purified anti-human CD45 antibody (BD Biosciences, cat. no. 555480); purified anti-human CD3 antibody (BioLegend, cat. no. 300416); purified anti-human p-STAT3 antibody (BD Biosciences, cat. no. 612357); purified anti-human p-histone H3 antibody (BioLegend, cat. no. 650802); purified anti-human IFN-γ antibody (BD Biosciences, cat. no. 554698); or purified anti-mouse MHC class II antibody (BioLegend, cat. no. 107637). We provide a list of antibodies used in CyTOF assays in Supplementary Table 1. Store according to the manufacturer’s instructions. ▲ CRITICAL The antibodies must be free of cysteine-containing carrier proteins (e.g., BSA) for efficient conjugation, as the carrier may compete for maleimide-functionalized groups. If a carrier is present, antibody cleanup kits (e.g., Abcam BSA Removal Kit, cat. no. ab173231; Melon Gel IgG Spin Purification Kit, Thermo Fisher Scientific, cat. no. 45206) can be used for antibody purification.

Cell assays

• Jurkat Clone E6–1 human T lymphocyte cell line (ATCC, cat. no. TIB-152). Human and mouse PBMCs are isolated from whole blood using Ficoll-Paque density gradient centrifugation. Cell suspensions from tissues or organs are prepared as previously described^9,39. For a reliable CyTOF analysis, at least 1.0 × 10⁶ cells per sample and two replicates are required. ! CAUTION The cell lines used in your research should be regularly checked to ensure that they are authentic and are not infected with mycoplasma. ! CAUTION All experiments involving animal or human samples should adhere to the relevant governmental and institutional ethics guidelines and regulations. Informed consent should be obtained from donors of human blood or tissue. All of our animal studies were performed in accordance with the investigators’ protocols approved by the Stanford University institutional animal care and use committee.

• RPMI 1640 cell culture medium (Life Technologies, cat. no. 21870–092)

• FBS (Thermo Fisher Scientific, cat. no. SH3007003)

• Penicillin-streptomycin (10,000 U/mL; Thermo Fisher Scientific, cat. no. 15140122)

• L-glutamine (100x; Thermo Fisher Scientific, cat. no. 25030081)

• Recombinant human IL-10 (BD Biosciences, cat. no. 554611)

• Recombinant human IL-12 (Peprotech, cat. no. 200–12)

• Recombinant human IL-18 (R&D Systems, cat. no. B001–5)

• Brefeldin A solution (1,000x; eBioscience, cat. no. 00-4506-51)

• Monensin solution (1,000x; eBioscience, cat. no. 00-4505-51)

• Ficoll-Paque plus density gradient medium (Thermo Fisher Scientific, cat. no. 45-001-749)

• Iridium Ir¹⁹¹/Ir¹⁹³ DNA intercalator (Fluidigm, cat. no. 201192A)

• 16% (wt/vol) Paraformaldehyde (PFA, electron microscopy grade; Electron Microscopy Sciences, cat. no. 15710) ! CAUTION PFA is an irritant. Avoid contact with skin and eyes. Avoid inhalation.

• Saponin (Sigma-Aldrich, cat. no. S-7900)

• Methanol (Thermo Fisher Scientific, cat. no. A412–4) ! CAUTION Methanol is flammable. Keep away from heat. Avoid contact with skin and eyes. Avoid inhalation.

• Dimethyl sulfoxide (DMSO; Sigma-Aldrich, cat. no. D2650)

• BSA fraction V (Sigma-Aldrich, cat. no. A-2153)

• Sodium azide (NaN₃; Sigma-Aldrich, cat. no. S-8032) ! CAUTION Sodium azide is highly toxic. Avoid contact with skin, eyes, and clothing. This compound is fatal if swallowed.

• Sodium phosphate dibasic heptahydrate (Sigma-Aldrich, cat. no. S9390)

• Potassium phosphate monobasic anhydrous (Sigma-Aldrich, cat. no. P0662)

• Potassium chloride (Thermo Fisher Scientific, cat. no. P330)

• Sodium chloride (Thermo Fisher Scientific, cat. no. S271)

Equipment

• Tabletop microcentrifuge (Eppendorf, model no. 5424)

• Tabletop refrigerated swinging-bucket centrifuge (Beckman Coulter, model no. Allegra 6R)

• Aspirator (VP Scientific, cat. no. VP 177A-1)

• Screw-top Eppendorf tubes (Thermo Fisher Scientific, cat. no. 02-707-353)

• Polystyrene round-bottom test tubes with 35-μ™ filter cap (FACS tubes; Thermo Fisher Scientific, cat. no. 08-771-23)

• Automated cell counter (Bio-Rad, model no. TC20)

• Digital-control water bath (Thermo Fisher Scientific, cat. no. 15-462-5Q)

• Mass cytometers (CyTOF version 1, CyTOF version 2, and CyTOF version 3 (Helios); Fluidigm)

• 3-kDa Amicon Ultra 0.5-mL centrifugal filter column (EMD Millipore, cat. no. UFC500396)

• 50-kDa Amicon Ultra 0.5-mL centrifugal filter column (EMD Millipore, cat. no. UFC505096)

• Syringe filter with 200-nm nylon membrane (Pall, cat. no. 4550)

Reagent setup

5.0% (vol/vol) HNO₃ solution

Mix 5 mL of high-purity concentrated HNO₃ with 95 mL of ddH₂O. Store for up to 2 years at RT. ! CAUTION HNO₃ is very corrosive. Avoid contact with skin, eyes, and clothing.

100 mM Ammonium acetate solution, pH 5.5

Dissolve 7.7 g of ammonium acetate in 995 mL of ddH₂O and add 1.03 mL of glacial acetic acid.

50 mM Lanthanide isotope solutions

Dissolve ~25 mg of lanthanide nitrate or chloride in an appropriate volume of L-buffer or in 100 mM ammonium acetate solution to obtain a 50 mM ^139–176Ln³⁺ solution. Mix gently by pipetting up and down using filter tips. Store at 4 °C for up to 2 years.

50 mM Yttrium solution

Dissolve ~10 mg of ⁸⁹Y(NO₃)₃•4H₂O in an appropriate volume of L-buffer or in 100 mM ammonium acetate solution to obtain a 50 mM ⁸⁹Y³⁺ solution. Mix gently by pipetting up and down using filter tips. Store at 4 °C for up to 2 years.

50 mM Indium isotope solutions

Dissolve ~10 mg of ¹¹³InCl₃ and ¹¹⁵InCl₃ individually in an appropriate volume of L-buffer or 100 mM ammonium acetate solution to obtain 50 mM ¹¹³In³⁺ and ¹¹⁵In³⁺ solutions. Mix gently by pipetting up and down using filter tips. Store at 4 °C for up to 2 years.

50 mM Bismuth solution

Dissolve ~25 mg of ²⁰⁹Bi(NO₃)₃•5H₂O in an appropriate volume of 5.0% (vol/vol) HNO₃ to obtain a 50 mM ²⁰⁹Bi³⁺ solution. Mix gently by pipetting up and down using filter tips. Store at 4 °C for up to 2 years. ▲ CRITICAL STEP The concentration of HNO₃ should not be <5.0% (vol/vol); a lower concentration of HNO₃ may result in precipitation of bismuth in the following steps.

10× PBS

Dissolve 320 g of sodium chloride, 8 g of potassium chloride, 46 g of sodium phosphate dibasic heptahydrate, 8 g of potassium phosphate monobasic (anhydrous) in 3 L of ddH₂O, adjust to pH 7.4 with sodium hydroxide, and bring to a volume of 4 L. Store at RT for up to 6 months. ! CAUTION Use high-purity reagents to avoid metal contamination from common lab chemicals.

PBS

Dilute 10x PBS at a 1:10 ratio with ddH₂O. Store at RT for up to 6 months.

Cell staining medium

Dissolve appropriate amounts of BSA and NaN3 to achieve concentrations of 0.5% (wt/vol) and 0.02% (wt/vol), respectively, in PBS. Store at 4 °C for up to 6 months.

Cell culture medium

Add 50 mL of FBS, 5 mL of penicillin-streptomycin, and 5 mL of L-glutamine to 500 mL of RPMI 1640 medium. Store at 4 °C for up to 6 months.

Iridium Ir¹⁹¹/Ir¹⁹³ DNA intercalator solution

Mix 1 mL of PBS with 100 μL of 16% (wt/vol) PFA solution. For methanol-permeabilized cells, add 0.25 μL of 500 μM iridium DNA intercalator (1:4,000); for non-permeabilized cells, add 0.40 μL of 500 μM iridium DNA intercalator (1:2,500). ! CAUTION Iridium compounds are highly toxic. Avoid contact with skin, eyes, and clothing.

4 mM TCEP solution for antibody partial reduction

Mix 8 μL of 500 mM TCEP stock with 992 μL of R-buffer; vortex briefly.

Antibody stabilization solution

Add 0.1% (vol/vol) NaN₃ to antibody stabilizer. Store it at 4 °C for up to 1 year.

Procedure

▲ CRITICAL The Procedure has two main parts: the first contains three sections describing three individual protocols for antibody conjugation; the second has one section describing cell staining procedures to validate and titrate the conjugated antibodies. Section 1 starts at Step 1 and describes the conjugation of reduced antibodies with Pd@mal-DOTA mass tags that are prepared as described in Box 1. Section 2 starts at Step 30 and describes the chelation of Y³⁺, In³⁺, or Ln³⁺ cations into Maxpar X8 polymers, and the conjugation with reduced antibodies. Section 3 starts at Step 63 and describes the chelation of Bi³⁺ cations into Maxpar X8 polymers, and the conjugation with reduced antibodies. Section 4 describes brief procedures and instructions for staining cells with different types of conjugated antibodies (i.e., against stable surface and intracellular molecules and against stimulation-induced signaling molecules and cytokines).

Section 1: conjugation of antibody with palladium isotopic mass tags

Partial reduction of antibody ● Timing ~1 h

• 1Add 100 μg of antibody stock to a 50-kDa 0.5-mL centrifugal filter column.

  ▲ CRITICAL STEP The filter columns are designed to hold up to 500 μL of solution. If 100 μg of antibody requires addition of >200 μL of solution, pre-concentrate the antibody first in the same filter column by spinning at 12,000g for 10 min at RT.

• 2Add 300 μL of R-buffer to the column to bring it a final volume of 500 μL.
• 3Spin the column at 12,000g for 10 min at RT. The final volume should not be >20 μL; discard the column flow-through.
• 4Add 100 μL of 4 mM TCEP solution to the antibody in the filter column, mix thoroughly by pipetting up and down using filter tips.
• 5Incubate the mixture at 37 °C for 30 min in a water bath.

  ▲ CRITICAL STEP The antibody should not be incubated in 4 mM TCEP solution for >30 min; a longer incubation may result in the reduction of disulfide bonds that are necessary for the structural integrity of the antibody.

• 6Add 300 μL of C-buffer to the partially reduced antibody in the filter column to stop the reaction.
• 7Spin the column at 12,000g for 10 min at RT. The final volume should not be >20 μL; discard the column flow-through.
• 8Add 400 μL of C-buffer to the partially reduced antibody in the filter column.
• 9Spin the column at 12,000g for 10 min at RT. The final volume should not be >20 μL; discard the column flow-through.

  ▲ CRITICAL STEP The second wash step is necessary to remove excess TCEP solution.

• 10Resuspend the partially reduced antibody in 100 μL of C-buffer. Mix by pipetting up and down using filter tips.

Conjugation of the reduced antibody with Pd@mal-DOTA mass tags ● Timing ~1 h

• 11Retrieve one PCR tube of 10 mM Pd@mal-DOTA mass tags (prepared as described in Box 1) with the desired isotope (¹⁰²Pd, ¹⁰⁴Pd, ¹⁰⁵Pd, ¹⁰⁶Pd, ¹⁰⁸Pd, or ¹¹⁰Pd).
• 12Warm the tube to RT. Vortex briefly and spin at 1,000g for 10 s in a microcentrifuge to ensure that the reagent is at the bottom of the tube.
• 13Add 1 μL of 10 mM Pd@mal-DOTA to the antibody solution (from Step 10).
• 14Mix the solution thoroughly by pipetting up and down using filter tips. Incubate the mixture at 37 °C for 1 h in a water bath.
• 15During the incubation, prepare three Zeba 7-kDa 0.5-mL spin desalting columns, remove the columns’ closures and loosen the caps. Place each column in a 2.0-mL collection tube.
• 16Spin at 1,500g for 1 min at RT to remove the storage solution.
• 17Make a mark on the side of each column where the compacted resin is slanted upward. Place the columns in the microcentrifuge with the mark facing outward in all subsequent centrifugation steps.
• 18Add 300 μL of W-buffer on top of the resin bed, spin at 1,500g for 1 min at RT, and discard the column flow-through.
• 19Add 300 μL of W-buffer on top of the resin bed, spin at 1,500g for 1 min at RT, and discard the column flow-through.
• 20Put these three columns into new 2.0-mL collection tubes marked W-1, W-2, and W-3, respectively.

Washing and recovering the Pd-tagged antibody ● Timing ~10 min

• 21Transfer the solution of conjugated antibody (from Step 14) to the top of the compact resin bed of the tube marked W-1.
• 22Spin at 1,500g for 2 min at RT to collect the flow-through. Discard the desalting column after use.
• 23Transfer the flow-through of W-1 to the top of the compact resin bed of the tube marked W-2.
• 24Spin at 1,500g for 2 min at RT to collect the flow-through. Discard the desalting column after use.
• 25Transfer the flow-through of W-2 to the top of the compact resin bed of the tube marked W-3.
• 26Spin at 1,500g for 2 min at RT to collect the flow-through. Discard the desalting column after use.
• 27Transfer the flow-through of W-3 to a new screw-top tube. Label it with the name of the antibody and the palladium isotope.

  ■ PAUSE POINT The conjugated antibody can be stored at 4 °C for up to 1 month.

  ? TROUBLESHOOTING

Quantification of the Pd-tagged antibody ● Timing ~15 min

• 28Determine the concentration of the Pd-tagged antibody using method A, which is described in Box 2.
• 29Dilute the Pd-tagged antibody to an appropriate concentration (0.2 mg/mL is recommended) in antibody stabilization solution.

  ■ PAUSE POINT Store at 4 °C for up to 6 months.

Section 2: conjugation of antibody with yttrium, indium, or lanthanide isotopes

Chelation of trivalent isotopic cations (Y³⁺, In³⁺, or Ln³⁺) into Maxpar X8 polymers ● Timing ~1 h

• 30Retrieve one tube (~200 μg) of lyophilized Maxpar X8 polymers, this is sufficient to conjugate 100 μg of IgG antibody. Spin the tube at 1,000g for 10 s at RT in a microcentrifuge to ensure that the reagent is at the bottom of the tube.
• 31Dissolve the polymers in 95 μL of L-buffer. Mix gently by pipetting up and down.
• 32Add 5 μL of 50 mM Y³⁺, In³⁺, or Ln³⁺ isotopic cations (in L-buffer or 100 mM ammonium acetate buffer, pH 5.5) to the polymer solution for a final concentration of 2.5 mM. Mix gently by pipetting up and down using filter tips.
• 33Incubate the mixture at RT for 1 h. Mix approximately every 20 min by pipetting up and down using filter tips.
• 34After 30 min, start Step 38 (partially reduce the IgG antibody) to ensure that the metal-isotope-chelated polymers and partially reduced antibody will be ready at the same time. If additional steps are required to pre-concentrate the antibody, this step should begin 10 min earlier.

  ▲ CRITICAL STEP The maleimide-functionalized groups of Maxpar X8 polymers hydrolyze in aqueous solutions; the reduction of the disulfide bonds of antibodies is reversible in aqueous solutions. To obtain efficient antibody conjugation, make sure that the chelated Maxpar X8 polymers and the reduced antibody are ready at same time.

Removal of free Y³⁺, In³⁺, or Ln³⁺ isotopic cations ● Timing ~30 min

• 35After 1-h incubation, add 300 μL of C-buffer to the isotope chelation mixture (from Step 33) for a final volume of 400 μL. Transfer the solution to a 3-kDa MWCO filter column.
• 36Spin the column at 14,000g for 30 min at RT. The final volume should not be >20 μL; discard the column flow-through.
• 37Re-suspend Y³⁺, In³⁺, or Ln³⁺ isotope-chelated polymers in 200 μL of C-buffer. Gently pipette to mix and rinse the walls of the filter column to increase the recovery of the polymers.

Partial reduction of the IgG antibody ● Timing ~1 h

• 38Add 100 μg of antibody stock to a 50-kDa 0.5-mL centrifugal filter column.

  ▲ CRITICAL STEP The filter columns are designed to hold up to 500 μL of solution. If 100 μg of antibody requires addition of >200 μL of solution, pre-concentrate the antibody first in the same filter column by spinning at 12,000g for 10 min at RT.

• 39Add 300 μL of R-buffer to the column to bring a final volume of 500 μL.
• 40Spin the column at 12,000g for 10 min at RT. The final volume should not be >20 μL; discard the column flow-through.
• 41Add 100 μL of 4 mM TCEP solution to the antibody in the filter column and mix thoroughly by pipetting up and down using filter tips.
• 42Incubate the mixture at 37 °C for 30 min in a water bath.

  ▲ CRITICAL STEP The antibody should not be incubated in 4 mM TCEP solution for >30 min; a longer incubation may result in the reduction of disulfide bonds that are necessary for the structural integrity of the antibody.

• 43Add 300 μL of C-buffer to the partially reduced antibody in the filter column to stop the reaction.
• 44Spin the column at 12,000g for 10 min at RT. The final volume should not be >20 μL; discard the column flow-through.
• 45Add 400 μL of C-buffer to the partially reduced antibody in the filter column.
• 46Spin the column at 12,000g for 10 min at RT. The final volume should not be >20 μL; discard the column flow-through.

  ▲ CRITICAL STEP The second wash step is necessary to remove excess TCEP solution.

Conjugation of the reduced antibody with metal-isotope-chelated polymers ● Timing ~1 h

• 47Transfer the isotope-chelated polymers suspended in C-buffer (from Step 37) to the partially reduced antibody in the 50-kDa MWCO filter column. Mix thoroughly by pipetting up and down using filter tips.
• 48Incubate the mixture at 37 °C for 1 h in a water bath.

Washing and recovering the metal-isotope-tagged antibody ● Timing ~50 min

• 49Add 200 μL of W-buffer to the filter column. Mix thoroughly by pipetting up and down using filter tips.
• 50Spin the filter column at 12,000g for 10 min at RT. The final volume should not be >20 μL; discard the column flow-through.
• 51Add 400 μL of W-buffer to the filter column. Mix thoroughly by pipetting up and down using filter tips.
• 52Spin the filter column at 12,000g for 10 min at RT. The final volume should not be >20 μL; discard the column flow-through.
• 53Repeat Steps 51 and 52 two more times.
• 54Add 50 μL of W-buffer to the filter column. Pipette to mix and rinse the walls of the filter thoroughly.
• 55Invert the filter column into a new collection tube.
• 56Spin the inverted filter and collection tube assembly at 2,000g for 2 min at RT.
• 57Gently remove the filter column from the collection tube. Add an additional 50 μL of W-buffer to the filter column. Pipette to mix and rinse the walls of the filter thoroughly.
• 58Invert the filter column into the same collection tube.
• 59Spin the assembly at 2,000g for 2 min at RT.
• 60Transfer the MitAb solution to a new screw-top tube.

  ■ PAUSE POINT The conjugated antibody can be stored at 4 °C for up to 1 month.

Quantification of the metal-isotope-tagged antibody ● Timing ~15 min

• 61Determine the concentration of Y-, In-, or Ln-tagged antibody using method A, which is described in Box 2.

  ? TROUBLESHOOTING

• 62Dilute the conjugated antibody to an appropriate concentration (0.2 mg/mL is recommended) in antibody stabilization solution.

  ▲ CRITICAL STEP Ideally, the antibody should be diluted such that 1–2 μL is sufficient for a 100-μL cell-staining reaction. This facilitates the creation of low-volume staining cocktails.

  ■ PAUSE POINT Store at 4 °C for up to 6 months.

Section 3: conjugation of antibody with bismuth

Chelation of ²⁰⁹Bi³⁺ cations into Maxpar X8 polymers ● Timing ~1 h

• 63Retrieve one tube (~200 μg) of lyophilized Maxpar X8 polymers; this is sufficient to conjugate 100 μg of IgG antibody. Spin at 1,000g for 10 s at RT in a microcentrifuge to ensure that the reagent is at the bottom of the tube.
• 64Dissolve the polymers in 95 μL of 5.0% (vol/vol) HNO₃. Mix gently by pipetting up and down.

  ▲ CRITICAL STEP Do not dissolve the polymers in other solutions such as ddH₂O or L-buffer because these solvents will result in the precipitation of ²⁰⁹Bi³⁺ cations.

• 65Add 5 μL of 50 mM ²⁰⁹Bi³⁺ solution (in 5.0% (vol/vol) HNO₃) to the polymer solution. The final concentration of ²⁰⁹Bi³⁺ cations will be 2.5 mM. Gently mix by pipetting up and down using filter tips.
• 66Incubate the mixture at RT for 1 h. Mix approximately every 20 min by pipetting up and down using filter tips.

Removal of free ²⁰⁹Bi³+ cations ● Timing ~1 h

• 67Add 300 μL of ddH₂O to the bismuth chelation mixture for a final volume of 400 μL. Transfer the solution to a 3-kDa MWCO filter column.

  ▲ CRITICAL STEP Do not add other solutions, such as PBS, R-buffer, or C-buffer, because these solvents may result in the precipitation of free ²⁰⁹Bi³⁺ cations.

• 68Spin the filter column at 14,000g for 30 min at RT. The final volume should not be >20 μL; discard the column flow-through.
• 69When centrifugation starts, begin Step 73 (partial reduction of IgG antibody) to ensure that ²⁰⁹Bi-chelated polymers and reduced antibody will be ready at the same time. If additional steps are required to pre-concentrate the antibody, this step should begin 10 min earlier.

  ▲ CRITICAL STEP The maleimide-functionalized groups of Maxpar X8 polymers hydrolyze in aqueous solutions; the reduction of the disulfide bonds of the antibodies is reversible in aqueous solutions. To obtain efficient antibody conjugation, make sure the chelated Maxpar X8 polymers and the reduced antibody are ready at same time.

• 70Add 400 μL of C-buffer to the filter column (from Step 68).

  ? TROUBLESHOOTING

• 71Spin the filter column at 14,000g for 30 min at RT. The final volume should not be >20 μL; discard the column flow-through.

  ▲ CRITICAL STEP The second wash in C-buffer is important for removing the excess ²⁰⁹Bi³⁺ cations and adjusting the pH value for antibody conjugation.

• 72Resuspend the ²⁰⁹Bi-chelated polymers in 200 μL of C-buffer. Gently pipette to mix and rinse the walls of the filter column to increase the recovery of the polymers.

Partial reduction of the IgG antibody ● Timing ~1 h

• 73Add 100 μg of antibody stock to a 50-kDa 0.5-mL centrifugal filter column.

  ▲ CRITICAL STEP The filter columns are designed to hold up to 500 μL of solution. If 100 μg of antibody requires addition of >200 μL of solution, pre-concentrate the antibody first in the same filter column by spinning at 12,000g for 10 min at RT.

• 74Add 300 μL of R-buffer to the column to bring it a final volume of 500 μL.
• 75Spin at 12,000g for 10 min at RT. The final volume should not be >20 μL; discard the column flow-through.
• 76Add 100 μL of 4 mM TCEP solution to the antibody in the filter column and mix thoroughly by pipetting up and down using filter tips.
• 77Incubate the mixture at 37 °C for 30 min in a water bath.

  ▲ CRITICAL STEP The antibody should not be incubated in 4 mM TCEP solution for >30 min; a longer incubation may result in the reduction of the disulfide bonds that are necessary for the structural integrity of the antibody.

• 78Add 300 μL of C-buffer to the partially reduced antibody in the filter column to stop the reaction.
• 79Spin at 12,000g for 10 min at RT. The final volume should not be >20 μL; discard the column flow-through.
• 80Add 400 μL of C-buffer to the partially reduced antibody in the filter column.
• 81Spin at 12,000g for 10 min at RT. The final volume should not be >20 μL; discard the column flow-through.

  ▲ CRITICAL STEP The second wash step is necessary to remove excess TCEP solution.

Conjugation of the reduced antibody with ²⁰⁹Bi-chelated polymers ● Timing ~1 h

• 82Transfer the ²⁰⁹Bi-chelated polymers suspended in C-buffer (from Step 72) to the partially reduced antibody in the 50-kDa MWCO filter column. Mix thoroughly by pipetting up and down using filter tips.
• 83Incubate the mixture at 37 °C for 1 h in a water bath.

Washing and recovering the ²⁰⁹Bi-tagged antibody ● Timing ~50 min

• 84Add 200 μL of W-buffer to the filter column. Mix thoroughly by pipetting up and down using filter tips.
• 85Spin the filter column at 12,000g for 10 min at RT. The final volume should not be >20 μL; discard the column flow-through.
• 86Add 400 μL of W-buffer to the filter column. Mix thoroughly by pipetting up and down using filter tips.
• 87Spin the filter column at 12,000g for 10 min at RT. The final volume should not be >20 μL; discard the column flow-through.
• 88Repeat Steps 86 and 87 two more times.
• 89Add 50 μL of W-buffer to the filter column. Pipette to mix and rinse the walls of the filter thoroughly.
• 90Invert the filter column into a new collection tube.
• 91Spin the inverted filter and collection tube assembly at 2,000g for 2 min at RT.
• 92Gently remove the filter column from collection tube. Add an additional 50 μL of W-buffer to the filter column. Pipette to mix and rinse the walls of the filter thoroughly.
• 93Invert the filter column into the same collection tube.
• 94Spin the assembly at 2,000g for 2 min at RT.
• 95Transfer the solution of ²⁰⁹Bi-tagged antibody to a new screw-top tube.

  ■ PAUSE POINT The conjugated antibody can be stored at 4 °C for up to 1 month.

Quantification of the ²⁰⁹Bi-tagged antibody ● Timing ~45 min

• 96Determine the concentration of ²⁰⁹Bi-tagged antibody using the method B, described in Box 2.
• 97Dilute the ²⁰⁹Bi-tagged antibody to an appropriate concentration (0.2 mg/mL is recommended) in antibody stabilization solution.

  ■ PAUSE POINT Store at 4 °C for up to 6 months.

Part 4

Cell staining for validation and titration of metal-isotope-tagged antibodies ● Timing ~45 min

• 98
  Identify the positive- and negative-control cell populations for the conjugated antibody. The differential selection of populations and stimulation procedures are described below: option A is for antibodies against stable surface or intracellular molecules (e.g., CD45, CD3, MHC II, and p-histone H3); option B is for antibodies against stimulation-inducible intracellular molecules or modifications (e.g., pSTAT3); and option C is for antibodies against secreted molecules (e.g., IFN-γ).

  1. Antibodies against a stable surface or intracellular molecule

     1. Add 1 mL of cell culture medium or cell staining medium at 37 °C to FACS tubes.
     2. For live-cell barcoding with Pd-tagged CD45 antibodies (Pd-CD45), 1.0 × 10⁶ Jurkat cells are added to each FACS tube and pelleted by centrifuging at 500g for 5 min at RT. In the strategy of 6-choose-1, six FACS tubes should be prepared, each containing one antibody of 100 μL of 2 μg/ml Pd-CD45; in the strategy of 6-choose-2, three FACS tubes should be prepared, each containing two antibodies of 50 μL of 2 μg/ml Pd-CD45; in the strategy of 6-choose-3, one FACS tube should be prepared containing three antibodies of 50 μL of 1.5 μg/ml Pd-CD45.
     3. Incubate the cells for 30 min at RT with gentle agitation.
     4. Wash the cells twice using 4 mL of cell staining medium by centrifuging at 500g for 5 min at RT.
  2. Antibody against an induced intracellular molecule (e.g., pSTAT3)

     1. Add 1 mL of the cell culture medium at 37 °C to FACS tubes labeled ‘stimulated’ and ‘unstimulated’. To obtain reliable results, at least two replicates are required.
     2. Add 10 × 10⁶ human PBMCs to each tube.
     3. Add stimulation molecules such as IL-10 to the tubes labeled ‘stimulated’ to a final concentration of 100 ng/mL; add an equal volume of cell culture medium to the tubes labeled ‘unstimulated’.
     4. Incubate the cells at 37 °C for 15 min.
        ▲ CRITICAL STEP Cellular-signaling events are very time sensitive. Be ready to proceed to Step 99 immediately.
        ▲ CRITICAL STEP The stimulation should result in selective induction of the activated form of the signaling molecule of interest. Additional information on antibody targets and appropriate stimulation is provided in previous work⁷⁶.
  3. Antibody against a secreted intracellular cytokine (e.g., IFN-γ)

     1. Add 1 mL of cell culture medium at 37 °C to FACS tubes labeled ‘stimulated’ and ‘unstimulated’. To obtain reliable results, at least two replicates are required.
     2. Add 10 × 10⁶ human PBMCs to each tube.
     3. Add a stimulation cocktail, such as a combination of IL-12 and IL-18, to final concentrations of 50 ng/mL to the tubes labeled ‘stimulated’; add an equal volume of cell culture medium to the tubes labeled ‘unstimulated’.
     4. Add the secretion inhibitors Brefeldin A and Monensin to each tube; 1,000x stocks should be added at a dilution of 1:1,000.
     5. Incubate the cells at 37 °C for 15 min.
        ▲ CRITICAL STEP The stimulation should result in selective induction of the activated form of the molecule of interest. Additional information on secreted cytokines and appropriate stimulation can be found in previous work²¹.

Fixing and permeabilization of the cells ● Timing ~30 min

• 99Add 100 μL of 16% (wt/vol) PFA to each FACS tube and pipette up and down thoroughly. Incubate for 10 min at RT.
• 100Wash the cells twice using 4 mL of cell staining medium by centrifuging at 500g for 5 min at RT.
• 101Add 1 mL of −20 °C methanol to each of the disrupted cell pellets. Vortex to mix and incubate on ice for 10 min.

  ■ PAUSE POINT Store at −80 °C for up to 1 month.

Immune staining with the titrated antibodies ● Timing ~1 h

• 102For antibody titrations, wash the cells twice using 4 mL of cell staining medium by centrifuging at 500g for 5 min at RT.
• 103Aspirate the supernatant and leave the cells in 300 μL of residual volume, then divide the cells into six new FACS tubes.
• 104Prepare a six-step and twofold serial dilution of the conjugated antibodies. Add 50 μL of the diluted antibodies to each 50 μL of cell sample. The final titration concentrations of the conjugated antibodies range from 250 to 8,000 ng/mL.
• 105Incubate the cells for 30 min at RT with gentle agitation.
• 106Wash the cells twice using 4 mL of cell staining medium by centrifuging at 500g for 5 min at RT.

Staining of the cells in DNA intercalator solution ● Timing ~30 min

• 107Add 1 mL of Ir DNA intercalator solution to each FACS tube and mix thoroughly. Incubate for at least 20 min at RT.

  ■ PAUSE POINT Cells can be stored in DNA intercalator solution for up to 3 d at 4 °C before acquisition on a mass cytometer.

• 108Wash the cells twice using 4 mL of cell staining medium by centrifuging at 500g for 5 min at RT.
• 109Wash the cells using 4 mL of ddH₂O by centrifuging at 500g for 5 min at RT and then resuspend the cells in ddH₂O at a concentration of 1–2 × 10⁶ cells per mL.
• 110Filter the cells through a 35-μm filter cap and measure each tube using the mass cytometer (illustrated in Fig. 4c). Collect cell events at a rate of 500–1,000 cells per s.

Troubleshooting

Troubleshooting advice can be found in Table 2.

Table 2 |.

Troubleshooting table

Step	Problem	Possible reason	Solution
Box 1, step 12	Inefficient conjugation of mass tags to antibodies	Maleimide-functionalized groups lose activities	Minimize the number of freeze-thaw cycles that the palladium isotopic mass tags undergo
Box 2, step 8	Absorbance at 562 nm is variable or does not increase with antibody concentration	Working solution and antibody are not mixed well	Mix thoroughly by pipetting up and down, and make sure that all the liquid is at the bottom of tube; incubate with gentle shaking
27	Inefficient washing by desalting columns	Improper sample loading	Load the sample directly into the center of the resin bed and carefully touch the pipette tip to the resin to expel the antibody sample
61	Lower recovery of antibody conjugates (<50%)	Precipitation induced by excess metal cations or denaturation due to reduction	Ensure complete washes of the polymer and metal cations with post-centrifugation filter volumes of <20 μL. Ensure that reduction time is not >30 min
61	Higher recovery of antibody conjugates (>95%)	Carrier proteins	Check manufacturer specifications for carrier proteins (BSA, gelatin, or other protein stabilizer). Obtain carrier-free stock or purify the antibody away from the carrier
		Improper antibody starting amount	Measure the concentration of the starting antibody stock by A280; ensure that the conjugation amount is 100 μg
70	White flocculent precipitate observed	The pH exchange causes the precipitation of bismuth	Redissolve the precipitate in 5.0–10.0% (vol/vol) HNO3 solution. Mix by vigorous pipetting up and down. Filter the solution through a syringe filter with a 200-nm nylon membrane. Wash the polymers again in 0.5% (vol/vol) HNO3 solution again

Timing

Section 1, conjugation of antibody with palladium isotopic mass tags

Steps 1–10, partial reduction of the IgG antibody (Fig. 5a): ~1 h

Steps 11–20, conjugation of the reduced antibody with Pd@mal-DOTA mass tags: ~1 h

Steps 21–27, washing and recovering the Pd-tagged antibody: ~10 min

Steps 28 and 29, quantification of the Pd-tagged antibody: ~15 min

Section 2, conjugation of antibody with yttrium, indium, or lanthanide isotopes

Steps 30–34, chelation of Y3⁺, In3⁺, or Ln³⁺ isotopic cations to Maxpar X8 polymers (Fig. 5b): ~1 h

Steps 35–37, removal of the free Y³⁺, In³⁺, or Ln³⁺ isotopic cations: ~30 min

Steps 38–46, partial reduction of the IgG antibody: ~1 h

Steps 47 and 48, conjugation of the reduced antibody with metal-isotope-chelated polymer: ~1 h

Steps 49–60, washing and recovery of the MitAb: ~50 min

Steps 61 and 62, quantification of the MitAb: ~15 min

Section 3, conjugation of antibody with bismuth

Steps 63–66, chelation of ²⁰⁹Bi³⁺ cations to Maxpar X8 polymers (Fig. 5c): ~1 h

Steps 67–72, removal of the free ²⁰⁹Bi³⁺ cations: ~1 h

Steps 73–81, partial reduction of the IgG antibody: ~1 h

Steps 82 and 83, conjugation of the reduced antibody with ²⁰⁹Bi-chelated polymers: ~1 h

Steps 84–95, washing and recovery of the ²⁰⁹Bi-tagged antibody: ~50 min

Steps 96 and 97, quantification of the ²⁰⁹Bi-tagged antibody: ~45 min

Section 4, cell staining for validation and titration of metal-isotope-tagged antibodies

Step 98, preparation of the cell samples: ~45 min

Steps 99–101, fixing and permeabilization of the cells: ~30 min

Steps 102–106, immune staining with the titrated antibodies: ~1 h

Steps 107–110, staining of the cells in DNA intercalator solution: ~30 min

Box 1, preparation of palladium isotopic mass tags: 8 h

Box 2, quantification of metal-isotope-tagged antibodies: 1 h

Anticipated results

Characterization of palladium isotopic mass tags

Six Pd@mal-DOTA mass tags were synthesized and measured using ESI-MS, according to the procedures described in Box 1. The chemical formula of mal-DOTA is C₂₂H₃₄N₆O₉, and its theoretical MW is 526.23; therefore, the main mass peak at an m/z value of 525.2 in negative-ion ESI-MS was identified as representing the single-charge negative molecular ions [M-1]⁻ of mal-DOTA (Fig. 6a). In the Pd@mal-DOTA complexes, two hydrogen ions of mal-DOTA are replaced by the coordinated Pd²⁺ cation; thus, the expected mass for Pd@mal-DOTA is the sum of the atomic weight of the palladium isotope and the MW of mal-DOTA minus 3 Da (i.e., M-3 + Pd). For example, as shown in Fig. 6b, the main mass peak of ¹⁰²Pd@mal-DOTA is at the m/z value of 625.2, as expected for [M-3 + 102]⁻ (i.e., 526.2 − 3 + 102 = 625.2). The mass spectra of ¹⁰⁴Pd@mal-DOTA, ¹⁰⁵Pd@mal-DOTA, ¹⁰⁶Pd@mal-DOTA, ¹⁰⁸Pd@mal-DOTA, and ¹¹⁰Pd@mal-DOTA are shown in Fig. 6c–g, respectively.

Cell barcoding assays using Pd-tagged CD45 antibodies

Multiplexed barcoding of live Jurkat cells was carried out using CD45 antibodies conjugated with palladium isotopes as described in Section 1 of the Procedure. Three different barcoding strategies are demonstrated. For the 6-choose-1 strategy, Fig. 7a shows the CyTOF measurements as ‘rain dots’ of each barcode of Pd-tagged CD45 and biaxial contour plots comparing pairs of barcoding channels. These data indicate that each barcoded cell sample was effectively distinguished and that the double-channel-positive populations were rare. For the 6-choose-2 strategy, ¹⁰⁶Pd- and ¹⁰⁸Pd-tagged CD45, ¹⁰⁸Pd- and ¹¹⁰Pd-tagged CD45, or ¹⁰⁶Pd- and ¹¹⁰Pd-tagged CD45 were used to stain Jurkat cells. Both ‘rain dots’ and biaxial contours demonstrate that there are three expected double-positive populations, as shown in Fig. 7b. For the 6-choose-3 strategy, ¹⁰²Pd-, ¹⁰⁴Pd-, and ¹⁰⁵Pd-tagged CD45 antibodies were used to stain Jurkat cells. The expected triple-positive populations were observed, as shown in Fig. 7c. In principle, by using the 6-choose-1 strategy, six samples can be measured simultaneously. The strategies of 6-choose-2 and 6-choose-3 enable multiplexing of 15 and 20, respectively. It should be taken into consideration that, because of saturation of cell immune staining, the higher multiplexing will result in the lower signal intensity in each barcoding channel and consequentially might decrease the recovery of cell debarcoding.

Titrations of various ²⁰⁹Bi-tagged antibodies

Antibody titration is an important experiment use to validate and examine the specificity and affinity of the conjugated antibody. It is used to determine the optimal concentration of antibody for cell staining. As an example of how to do this, a wide variety of antibodies against stable surface and intracellular molecules and against stimulation-induced signaling molecules and cytokines have been successfully conjugated to bismuth mass tags, as described in Section 3 of the Procedure. Titration experiments were performed in positive- and negative-control cells to determine the maximum ratio of positive signal to background, which is referred to as the optimal concentration. As shown in Fig. 8a,²⁰⁹Bi-tagged MHC class II antibody was incubated with mouse PBMCs at concentrations ranging from 250 to 8,000 ng/mL in two fold dilutions. The titration curves were generated by analysis of B cells, which express MHC class II antigens, and T cells, which do not. Figure 8b,c show the titration histograms of intracellular phosphorylation antibodies of ²⁰⁹Bi-tagged phospho-STAT3(Tyr705) and ²⁰⁹Bi-tagged phospho-histone H3(Ser28) at their optimal concentrations. Figure 8d shows the titration histogram of the intracellular cytokine antibody of ²⁰⁹Bi-tagged IFN-γ at the optimal concentration in stimulated and unstimulated natural killer cells. Figure 8e shows the titration histogram of secondary antibody of ²⁰⁹Bi-tagged anti-PE at the optimal concentration in PE-conjugated CD3 antibody-stained T cells and B cells of human PBMCs. These results indicate that the bismuth conjugation protocol is suitable for various types of antibodies used in mass cytometry.

Comparisons of bismuth- and lanthanide-tagged antibodies in high-dimensional assays

By following the protocol described in Section 2 of the Procedure, a number of antibodies have been conjugated with yttrium, indium, and lanthanide isotopic mass tags (Supplementary Table 1). In high-dimensional CyTOF assays, bismuth- and lanthanide-tagged antibodies should provide comparable results without the isotope crosstalk. To demonstrate this, the same antibodies were conjugated with both bismuth and lanthanide mass tags: (i) human anti-CD3 tagged with ²⁰⁹Bi and ¹⁷⁰Er, (ii) human anti-CD56 tagged with ²⁰⁹Bi and ¹⁷⁶Yb, and (iii) mouse anti-MHC class II tagged with ²⁰⁹Bi and ¹⁷⁶Yb. Three separate comparisons were carried out in Jurkat cells, human PBMCs, and immune cells of mouse organs.

First, Jurkat cells were stained with lanthanide-tagged antibodies against CD19, CD4, CD11a, CD5, CD7, CD184, and CD2, and either ²⁰⁹Bi-tagged CD3 or ¹⁷⁰Er-tagged CD3. As the results in biaxial scatter plots (Fig. 9a, left) and radar charts (Fig. 9a, right) show, bismuth- and lanthanide-tagged CD3 provided comparable values of CD3 expression in Jurkat cells, and mean intensities of other compared CD markers had a strong correlation (Pearson correlation, r = 0.997, P < 0.00001, Supplementary Fig. 1a). Second, human PBMCs were then stained with 37 antibodies against surface markers and three intercellular markers (Supplementary Table 1), and either ²⁰⁹Bi-tagged CD56 or ¹⁷⁶Yb-tagged CD56. Fifteen immune cell subpopulations were manually gated (Supplementary Fig. 2). As the results in viSNE (visualization of t-distributed stochastic neighbor embedding) plots (Fig. 9b) and heat maps (Fig. 9c) show, there are highly comparable immunophenotypic clustering and strong correlations between data collected using ²⁰⁹Bi-tagged CD56 and ¹⁷⁶Yb-tagged CD56 (Pearson correlation, r = 0.991, P < 0.00001, Supplementary Fig. 1b). Third, comparison of ²⁰⁹Bi- and ¹⁷⁶Yb-tagged MHC class II antibodies in scaffold maps revealing the immune organization of bone marrow for C57BL/6 mice are shown in Fig. 10.