Protocol for live cell barcoding and immunophenotyping of human hematological malignancies using cytometry by time of flight

Summary

High-dimensional cell profiling by cytometry by time of flight (CyTOF) enables simultaneous characterization of rare cell populations but requires many antibodies and is sensitive to staining variability. Here, we present a workflow for immunophenotyping human hematological malignancy samples, reducing costs and minimizing technical variation. We describe steps for sample collection, live-cell barcoding using anti-β2-microglobulin and CD147 antibodies, cell staining, data collection, and subsequent analyses.

For complete details on the use and execution of this protocol, please refer to Mikami et al.¹

Graphical abstract

Highlights

• •Perform live-cell barcoding using anti-β2-microglobulin and CD147 antibodies
• •Enhance chemokine marker intensity by staining cells in culture medium at 37°C
• •Profile the immunological landscape of human hematological malignancies

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

High-dimensional cell profiling by cytometry by time of flight (CyTOF) enables simultaneous characterization of rare cell populations but requires many antibodies and is sensitive to staining variability. Here, we present a workflow for immunophenotyping human hematological malignancy samples, reducing costs and minimizing technical variation. We describe steps for sample collection, live-cell barcoding using anti-β2-microglobulin and CD147 antibodies, cell staining, data collection, and subsequent analyses.

Before you begin

The protocol below describes the specific steps for accessing immunophenotypes of human hematological malignancy samples, including peripheral blood and bone marrow aspirate, using CyTOF. We design the CyTOF panel primarily to evaluate T cell phenotypes; however, marker selection can be modified depending on the immune or tumor cell populations of interest.

To evaluate CD19 chimeric antigen receptor (CAR) expression on T cells, we include FMC63 (a single-chain variable fragment that binds to human CD19) as part of the CyTOF panel. Omit this marker if detection of CD19 CAR-T cells is not required.

When designing a CyTOF panel, users should be aware that signal spillover can occur in the mass channels M ± 1 (channel spreading), M + 16 (oxidation), and other isotope channels depending on the metal impurities in accordance with the particular metal.² These effects should be addressed during panel design by optimizing marker-metal assignments, avoiding placement of high-signal markers adjacent to channels expected to detect low-abundance signals.

Metal-labeled antibodies are either purchased pre-labeled from Standard BioTools or conjugated using Maxpar® labeling kits (X8 for lanthanides, and MCP9 for cadmium) according to the manufacturer’s instructions (https://standardbio.com/support/instrument-support/cytof-xt-system-support/#documents) except for cisplatin-labeled antibodies produced with own hands.³ The metal isotopes 113In, 115In, and 139La are not included in the labeling kit, therefore, these isotopes must be purchased separately, and prepared as 50 mM stock solutions dissolved in the L-buffer provided in the labeling kit. Self-conjugated (conjugated by Maxpar® labeling kits) antibodies are stored at a stock concentration of 0.5 mg/mL. All antibodies are titrated for optimal staining concentrations. To efficiently titrate the numerous CyTOF antibodies, it is important to titrate several antibodies together, taking into account the types of metal isotopes, leakage to adjacent channels, and oxidation.⁴

Innovation

This protocol enables high-dimensional immunophenotyping of valuable human hematological malignancy samples through live cell multiplexing while preserving physiological marker expression.

Compared with cell barcoding that requires fixation, live cell barcoding preserves physiological cell-surface marker expression. Anti-CD45 antibodies are commonly used to stain hematopoietic cells (excluding red blood cells and platelets). However, hematological malignant cells such as leukemic blasts often exhibit dim or reduced CD45 expression. Therefore, it is necessary for barcoding hematological malignancy samples to use markers which are expressed on both normal cells and malignant cells. β2-microglobulin (B2MG), a component of MHC class I molecules, is present on all nucleated cells, except for some tumor cells with reduced MHC class I expression due to immune evasion.⁵ In contrast, CD147 (basigin; BSG, extracellular matrix metalloproteinase inducer; EMMPRIN) is frequently overexpressed in various hematological malignancies and solid tumors.⁶ Accordingly, we use B2MG and CD147 as barcoding markers that can be applied to a wide range of hematological malignancy samples. In addition, the metal combinations used for barcoding were designed so that samples could be separated diagonally. This allows for improved sample discrimination and more efficient removal of doublets.

After the barcoding step, chemokine receptor markers are stained by reacting at 37°C while the cells are still viable.^7,8,9 Since chemokine receptors move between the cell membrane surface and the cytoplasm, keeping cell active through this type of incubation results in stronger staining of chemokine receptors. This protocol can be applied to immune profiling of other tumor samples as well as hematological malignancy samples with prior validation.

Institutional permissions

The study was approved by the Kyoto University Hospital Ethical Board (approval number: G-1030) and related institutions. All patients or their guardians provided informed consent for human sample collection. The study was conducted in accordance with ethics guidelines, including the Declaration of Helsinki.

Sample processing and preservation

Timing: 1 h

• 1.
  Mononuclear cell (MNC) isolation from whole blood or bone marrow aspirates by density gradient centrifugation.

  • a.
    Collect blood or bone marrow aspirates using ethylenediaminetetraacetic acid (EDTA) or heparin as an anticoagulant.

    Note: While EDTA is preferred for maintaining cell morphology and general surface marker staining, heparin is recommended for high-precision intracellular signal analysis (such as phosphorylation analysis).

  • b.
    Mix Lymphoprep™ thoroughly before use by inverting the bottle several times.
  • c.
    Add Lymphoprep™ to tube.
  • d.
    Dilute samples with an equal amount of complete RPMI (cRPMI) and layer them on top of Lymphoprep™, being careful to minimize mixing diluted samples with Lymphoprep™.
  • e.
    Centrifuge at 400× g for 30 min at 15°C–25°C with the brake off.
  • f.
    Collect MNCs at the plasma: Lymphoprep™ interface without disturbing erythrocyte/granulocyte pellet.
  • g.
    Wash MNCs once with cRPMI, by centrifugation for 5 min at 400× g, 4°C. Remove supernatant.
• 2.
  Cell count and preservation.

  • a.
    Resuspend cells with cRPMI and count cells using a hemocytometer or Burker camera.
  • b.
    Spin down the cells by centrifugation for 5 min at 400× g, 4°C. Remove supernatant.
  • c.
    Add 1 mL of CELLBANKER® 1 per 5 × 10⁵–5 × 10⁶ cells and gently mix it with pipetting.
  • d.
    Dispense the cell suspension into 1.5 mL cryotubes.
  • e.
    Put cryotubes in CoolCell® LX and freeze at −80°C.

Note: Samples should be stored in liquid nitrogen for a long period of time.

Sample thawing and cell number adjustment

Timing: 1 h

• 3.
  Sample thawing. Troubleshooting 1.

  • a.
    Prepare a water bath and prewarm cRPMI at 37 °C.
  • b.
    Thaw samples quickly by gently shaking cryotubes in a 37°C water bath.

    CRITICAL: Remove tubes from the water bath when the cell suspension has incompletely dissolved and become slushy. This step should take less than 2 min.

  • c.
    Work fast but carefully pipette thawed samples from the cryovial into a 15 mL tube.
  • d.
    Wash the vial with 1mL of prewarmed cRPMI and slowly add to the tube. Repeat this operation once more.
  • e.
    Add 8 mL of warm cRPMI to the tube.
  • f.
    Centrifugate the tube at 400× g for 5min, at 15°C–25°C. Decant supernatant.
  • g.
    Add cell staining medium (CSM) to resuspend the pellet.
• 4.Determine the cell number using a hemocytometer or Burker camera.

Note: In this step you can check cell viability.

• 5.Adjust the cell number in each tube to be approximately the same.
• 6.Centrifugate the tube (at 400× g for 5min, at 15°C–25°C) and remove the supernatant by careful aspiration using a pipette, leaving approximately 30 μL of final volume.

Note: For immunophenotyping, the minimum recommended cell number is 1 × 10⁶ cells/sample.

Note: Either proceed to the next step quickly or place on ice if many samples are being processed.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
89Y-anti human CD45 dilution 1:100	Standard BioTools	Cat#3089003B, RRID:AB_2938863
141Pr-anti human CD196 (CCR6) dilution 1:100	Standard BioTools	Cat#3141003A, RRID:AB_2687639
142Nd-anti-human CD19 dilution 1:100	Standard BioTools	Cat#3142001B, RRID:AB_3661857
143Nd-anti-human HLA-DR dilution 1:200	Standard BioTools	Cat#3143013B, RRID:AB_3661844
144Nd-anti-human CD38 dilution 1:100	Standard BioTools	Cat#3144014B, RRID:AB_2687640
145Nd-anti-human CD4 dilution 1:100	Standard BioTools	Cat#3145001B, RRID:AB_3661845
146Nd-anti-human CD8a dilution 1:50	Standard BioTools	Cat#3146001B, RRID:AB_3661846
148Nd-anti-human CD274 (PD-L1) dilution 1:100	Standard BioTools	Cat#3148017B, RRID:AB_3677831
149Sm-anti-human CD25 dilution 1:100	Standard BioTools	Cat#3149010B, RRID:AB_2756416
150Nd-anti-human CD134 (OX40) dilution 1:25	Standard BioTools	Cat#3150023B, RRID:AB_2938869
152Sm-anti-human TCRγδ dilution 1:25	Standard BioTools	Cat#3152008B, RRID:AB_2687643
154Sm-anti-human CD3 dilution 1:100	Standard BioTools	Cat#3154003B, RRID:AB_2811086
155Gd-anti-human CD27 dilution 1:200	Standard BioTools	Cat#3155001B, RRID:AB_2687645
156Gd-anti-human CD183 (CXCR3) dilution 1:100	Standard BioTools	Cat#3156004B, RRID:AB_2687646
158Gd-anti-human CD10 dilution 1:100	Standard BioTools	Cat#3158011B, RRID:AB_2921314
163Dy-anti-human CD56 dilution 1:200	Standard BioTools	Cat#3163007B, RRID:AB_3096015
164Dy-anti-human CD161 dilution 1:100	Standard BioTools	Cat#3164009B, RRID:AB_2687651
166Er-anti-human CD34 dilution 1:100	Standard BioTools	Cat#3166012B, RRID:AB_2756424
167Er-anti-human CD197 (CCR7) dilution 1:100	Standard BioTools	Cat#3167009A, RRID:AB_2858236
170Er-anti-human CD45RA dilution 1:400	Standard BioTools	Cat#3170010B, RRID:AB_2938862
171Yb-anti-human CD185 (CXCR5) dilution 1:100	Standard BioTools	Cat#3171014B, RRID:AB_2858239
174Yb-anti-human CD279 (PD-1) dilution 1:50	Standard BioTools	Cat#3174020B, RRID:AB_2868402
175Lu-anti-human CD194 (CCR4) dilution 1:50	Standard BioTools	Cat#3175035A, RRID:AB_2921320
176Yb-anti-human CD127 dilution 1:200	Standard BioTools	Cat#3176004B, RRID:AB_3665122
209Bi-anti-human CD11b dilution 1:200	Standard BioTools	Cat#3209003B, RRID:AB_2687654
Purified-anti-human TCRαβ dilution 1:25	BioLegend	Cat#306702, RRID:AB_314628
Purified-anti-human CD33 dilution 1:100	BioLegend	Cat#303402, RRID:AB_314346
Purified-anti-human CD14 dilution 1:25	BioLegend	Cat#301802, RRID:AB_314184
Purified-anti-human CD147 dilution 1:100	BioLegend	Cat#306202, RRID:AB_314586
Purified-anti-human β2 microglobulin dilution 1:100	BioLegend	Cat#316302, RRID:AB_492835
Biotin CAR (FMC63) dilution 1:12.5	ACROBiosystems	Cat#FM3-BY54
Purified anti Biotin dilution 1:25	BioLegend	Cat#409002, RRID:AB_10642032
Purified-anti-human CD39 dilution 1:12.5	BioLegend	Cat#328202, RRID:AB_940438
Purified-anti-human ICOS dilution 1:100	BioLegend	Cat#313502, RRID:AB_416326
Purified-anti-human CD57 dilution 1:400	BioLegend	Cat#359602, RRID:AB_2562403
Purified-anti-human CD152 (CTLA-4) dilution 1:100	BioLegend	Cat#349902, RRID:AB_10642827
Purified-anti-human CD73 dilution 1:50	BioLegend	Cat#344002, RRID:AB_2154067
Purified-anti-human CD22 dilution 1:100	BioLegend	Cat#302502, RRID:AB_314264
Purified-anti-human Foxp3 dilution 1:25	eBioscience	Cat#14-4777-82, RRID:AB_467556
Purified-anti-human Vα24 dilution 1:12.5	BioLegend	Cat#342902, RRID:AB_2229301
Purified-anti-human CD195 (CCR5) dilution 1:50	BioLegend	Cat#359102, RRID:AB_2562457
Purified-anti-human CD16 dilution 1:100	BioLegend	Cat#302002, RRID:AB_314202
Purified-anti-human Tim-3 dilution 1:50	BioLegend	Cat#345002, RRID:AB_2116574
Purified-anti-human TIGIT dilution 1:25	BioLegend	Cat#372702, RRID:AB_2632714
Biological samples
Human bone marrow and blood samples, infusion products (tisagenlecleucel)	Kyoto University Hospital	N/A
Chemicals, peptides, and recombinant proteins
Cell-ID™ IdU	Standard BioTools	Cat#201127
Maxpar® MCP9 Antibody Labeling Kit, 111Cd	Standard BioTools	Cat#201111A
Maxpar® MCP9 Antibody Labeling Kit, 112Cd	Standard BioTools	Cat#201112A
Maxpar® MCP9 Antibody Labeling Kit, 114Cd	Standard BioTools	Cat#201114A
Maxpar® MCP9 Antibody Labeling Kit, 116Cd	Standard BioTools	Cat#201116A
Maxpar® X8 Antibody Labeling Kit, 147Sm	Standard BioTools	Cat#201147A
Maxpar® X8 Antibody Labeling Kit, 151Eu	Standard BioTools	Cat#201151A
Maxpar® X8 Antibody Labeling Kit, 159Tb	Standard BioTools	Cat#201159A
Maxpar® X8 Antibody Labeling Kit, 160Gd	Standard BioTools	Cat#201160A
Maxpar® X8 Antibody Labeling Kit, 161Dy	Standard BioTools	Cat#201161A
Maxpar® X8 Antibody Labeling Kit, 162Dy	Standard BioTools	Cat#201162A
Maxpar® X8 Antibody Labeling Kit, 165Ho	Standard BioTools	Cat#201165A
Maxpar® X8 Antibody Labeling Kit, 168Er	Standard BioTools	Cat#201168A
Maxpar® X8 Antibody Labeling Kit, 169Tm	Standard BioTools	Cat#201169A
Maxpar® X8 Antibody Labeling Kit, 172Yb	Standard BioTools	Cat#201172A
Maxpar® X8 Antibody Labeling Kit, 173Yb	Standard BioTools	Cat#201173A
Cell-ID™ Cisplatin-194Pt-100 μL	Standard BioTools	Cat#201194
Cell-ID™ Cisplatin-195Pt-100 μL	Standard BioTools	Cat#201195
Cell-ID™ Cisplatin-196Pt-100 μL	Standard BioTools	Cat#201196
Cell-ID™ Cisplatin-198Pt-100 μL	Standard BioTools	Cat#201198
113In >93atom%	Trace Sciences International	Cat#In-113
115In >99atom%	Trace Sciences International	Cat#In-115
Lanthanum (III) chloride heptahydrate	Merck	Cat#203521
Cell-ID™ Intercalator-Rh	Standard BioTools	Cat#201103A
Cell Acquisition Solution	Standard BioTools	Cat#201240
EQ Four Element Calibration Beads	Standard BioTools	Cat#201078
Lymphoprep™	Alere Technologies	Cat#1858-1
CELLBANKER® 1	ZENOAQ RESOURCE	Cat#11910
Phosphate Buffered Saline (PBS)	Nacalai Tesque	Cat#14249-24
Ethylenediaminetetraacetic acid disodium salt (EDTA 2Na) dihydrate	Nacalai Tesque	Cat#15130-95
Sodium Azide (NaN3)	Nacalai Tesque	Cat#31208-82
RPMI-1640 (with L-Glutamine and Phenol Red)	FUJIFILM Wako	Cat#189-02025
Fetal bovine serum (FBS)	Gibco	Cat#10270-106
Penicillin-Streptomycin Solution (x100)	FUJIFILM Wako	Cat#168-23191
Pierce™ Universal Nuclease for Cell Lysis	Thermo Fisher Scientific	Cat#88702
Fc receptor Binding Inhibitor Functional Grade Monoclonal Antibody	eBioscience	Cat#16-9161-73
Dichloro-(ethylenediamine) (DCED) palladium (II)	Sigma-Aldrich	Cat#400076-1G
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	Cat#D2650-100ML
eBioscience™ Foxp3/Transcription Factor Staining Buffer Set	eBioscience	Cat#00-5523-00
Trypan Blue	Standard Vendors	N/A
Deposited data
CyTOF data	Mikami et al.	Zenodo: https://doi.org/10.5281/zenodo.13731333, https://doi.org/10.5281/zenodo.13731405
Software and algorithms
CyTOF® Software	Standard BioTools	Version 7.0.8493
Cytobank Premium	Beckman Coulter	https://premium.cytobank.org/cytobank/
FlowJo™	Becton, Dickinson and Company (BD)	Version 10.8.1
BioRender	BioRender	https://www.biorender.com/
Other
Helios The CyTOF® system	Standard BioTools	Cat#107001
AS ONE Thermax Water Bath TM-1A	AS ONE	Cat#1-4594-41
Countess™ 3 Automated Cell Counter	Thermo Fisher Scientific	Cat#AMQAX2000
Falcon® 15 mL conical tube	Corning	Cat#352196
Falcon® Cell Strainer tube (35 μm)	Corning	Cat#352235
CoolCell® LX	Biocision	Cat#BCS-405
CO2 incubator (humidified, 37°C, 5% CO2)	Standard Vendors	N/A
Biological safety cabinet	Standard Vendors	N/A
Microscope	Standard Vendors	N/A
4°C Fridge	Standard Vendors	N/A
Centrifuge (suitable for 15 mL tubes with 4°C option)	Standard Vendors	N/A
Minicentrifuge	Standard Vendors	N/A
Neubauer chamber	Standard Vendors	N/A
1.5 mL tube	Standard Vendors	N/A
Pipettes (P1000, P200, P20, P2)	Standard Vendors	N/A
Filtered pipette tips (P1000, P200, P20, P2)	Standard Vendors	N/A

Materials and equipment

CyPBS: PBS which is confirmed to have low barium and other metals used for CyTOF. Store at 4°C and use it within 12 months.

Alternatives: It is possible to use Maxpar® PBS instead of CyPBS.

Note: Metal contaminants such as barium and lead could be present in soaps, tap water and laboratory glassware. To avoid metal contamination, single-use, non-autoclaved plasticware is recommended at all CyTOF staining steps.

Palladium stock

Reagent	Final concentration	Amount
DECD-palladium	250 μM	0.07125 mg
DMSO	N/A	1.2 mL
Total	N/A	1.2 mL

Store at −20°C and use it within 5 years.

Note: Dissolve 9.5 mg of DCED-palladium powder in 4 ml of DMSO (10 mM palladium concentration). The tube containing this solution is capped and placed in an incubator at 37°C for 48 h for preconditioning to reduce cytotoxicity. Then, dilute 30 μL of the solution with 1170 μL of DMSO to make 1200 μL of a 250 μM stock solution. Divide each 2.1 μL into tubes.

Complete RPMI (cRPMI)

Reagent	Final concentration	Amount
RPMI-1640	N/A	445 mL
FBS	10%	50 mL
Penicillin-Streptomycin Solution (x100)	1%	5 mL
Total	N/A	500 mL

Store at 4°C and use it within 12 months.

CRITICAL: cRPMI should be prepared in sterile conditions in a laminar flow hood.

Cell Staining Medium (CSM)

Reagent	Final concentration	Amount
CyPBS	N/A	499.5 mL
FBS	0.1%	0.5 mL
EDTA 2Na dihydrate	2 mM	372 mg
Sodium azide (NaN3)	0.01%	50 mg
Total	N/A	500 mL

Store at 4°C and use it within 12 months.

Alternatives: It is possible to use Maxpar® Cell Staining Buffer instead of CSM.

CRITICAL: Do not sterilize CyPBS and CSM by autoclaving. Autoclaving can induce metal contamination of the buffers.

Step-by-step method details

Live-cell barcoding utilizing B2MG and CD147

Timing: 1 h 30 min

This step describes live cell barcoding procedure combining anti-B2MG and anti-CD147 antibodies. The barcoding system stains all hematopoietic cells including malignant tumors, and allows for the separation of cellular data for each sample after data acquisition.

CRITICAL: Since metal-containing reagents are toxic or harmful to the environment, they should be handled with care in accordance with their toxicity profiles and disposed of as recommended by local authorities.

Note: The following amounts of barcoding antibodies are for staining 3 × 10⁵–2.5 × 10⁶ cells/sample. If the number of cells per sample is larger or smaller, adjust the amount of the antibody reaction solution accordingly.

• 1.
  Preparation of mixtures of barcoding antibodies in each metal isotope.

  • a.
    For each metal isotope, mix equal amounts of anti-B2MG and anti-CD147 antibodies.

Note: Make this antibody mixture slightly more than you need. For example, if you stain 8 samples, mix 4.5 μL each of B2MG and CD147 antibodies (total 9 μL) for each metal isotope.

• 2.
  Live cell barcoding.

  • a.
    Thoroughly resuspend each sample in 50 μL of CSM by pipetting.
  • b.
    Add barcodes referring to the predetermined combinations (Table 1) to each sample.

    Note: A six-choose-three (six metals in combinations of three) with a potential for 20 combinations is shown as an example. However, six-choose-one and six-choose-two are viable alternatives for up to 6 or 15 samples respectively and reduce reagent consumption. However, the combination shown above is optimal for performing barcode removal using a diagonal arrangement, as will be discussed later.

  • c.
    After adding the three metal barcodes to each sample, mix quickly by tapping.
  • d.
    Incubate for 30 min at 15°C–25°C.
  • e.
    Wash twice with 2 mL CSM, by centrifugation for 5min at 400×g, at 15°C–25°C.
  • f.
    Resuspend each barcoded sample in 0.5 mL cRPMI and mix the samples together by pipette before centrifuging for 10 min at 400× g, at 15°C–25°C.
  • g.
    Remove the supernatant by careful aspiration using a pipette, leaving approximately 30 μL of final volume.

Table 1.

Combination of metal labels for live cell barcoding

Sample No.	114Cd	116Cd	194Pt	195Pt	196Pt	198Pt
1	–	+	–	–	+	+
2	+	–	–	–	+	+
3	–	+	+	–	+	–
4	+	–	+	–	+	–
5	–	+	–	+	–	+
6	+	–	–	+	–	+
7	–	+	+	+	–	–
8	+	–	+	+	–	–

+ = Add 2 μL of metal conjugated antibodies (B2MG 1 μL+ CD147 1 μL).

Multi-step cell staining

Timing: 4 h

In this step, cells are stained sequentially for chemokine receptor markers, cell surface markers, and intracellular markers. Chemokine receptor markers can be stained more strongly, improving the resolution of the location of cell populations, by keeping cells viable at 37°C incubation.

Note: The following antibody panels are designed for staining 1 × 10⁶–1 × 10⁷ cells of mixed samples after barcoding. If the number of cells is larger or smaller, adjust the amount of the antibody reaction solution accordingly.

• 3.
  Fc blocking (+ CAR staining).

  • a.
    Resuspend the sample in 10 μL CyPBS and add 10 μL of Fc block, mix quickly by tapping and incubate for 10 min at 15°C–25°C.
  • b.
    Add 4 μL of an anti-FMC63 antibody, mix quickly by tapping and incubate for 10 min at 15°C–25°C.

Note: An anti-FMC63 antibody is added for detecting CD19 CAR in the original research.¹ If CAR detection is not required, skip this step.

• 4.
  Chemokine receptor marker stain.

  • a.
    Add chemokine receptor marker stain cocktail (Table 2), mix well with pipette and incubate 30 min at 37°C water bath.
  • b.
    Wash twice with 2 ml CSM, by centrifugation for 5 min at 400× g, at 15°C–25°C.
  • c.
    Remove the supernatant by careful aspiration using a pipette, leaving approximately 30 μL of final volume.
• 5.
  Cell surface marker stain and dead cell stain.

  • a.
    Add cell surface marker stain cocktail (Table 2), mix well with pipette and incubate 30 min at 15°C–25°C.
  • b.
    Wash twice with 2 ml CyPBS, by centrifugation for 5 min at 400× g, at 15°C–25°C. Remove the supernatant by decantation.
  • c.
    Prepare 1 mL of 500 nM DCED palladium solution. To make this, mix 2 μL of 250 μM palladium stock with 1 mL CyPBS.
  • d.
    Add the palladium solution, mix and incubate for 10 min at 15°C–25°C.
  • e.
    Wash with 2 ml CSM, by centrifugation for 5 min at 400× g, 4°C. Remove the supernatant by decantation.
• 6.
  Intracellular marker stain and DNA stain.

  • a.
    To fix and permeabilize cells, add 1 mL of eBioscience™ fix/perm buffer, mix with pipette and incubate for 45 min at 4°C.

    Note: Fix/perm buffer is a ratio of 1:3 of fix/perm concentrate to fix/perm diluent (250 μL + 750 μL).

  • b.
    Wash three times with 2 mL 1× perm buffer by centrifugation at 800× g for 5min, 4°C.

    Pause point: Samples can be stored at this point for up to 48 h at 4°C.

    Note: 1× perm buffer is a diluent of 10× perm buffer with Milli-Q (Make 25 mL: 2.5 mL of 10× stock + 22.5 mL of Milli-Q).

  • c.
    Remove the supernatant by careful aspiration using a pipette, leaving approximately 50 μL of final volume.
  • d.
    Add intracellular marker stain cocktail (Table 2), mix well with pipette and incubate 30 min at 4°C.
  • e.
    Wash twice with 2 mL of 1× perm buffer and then once with 2 mL of CyPBS (centrifuge at 800× g for 5 min at 4°C).
  • f.
    Prepare 1 mL of 2% formaldehyde (FA) with 1:500 Cell-ID Intercalator Rhodium: 125 μL of 16% FA, 875 μL of CyPBS, and 2 μL of Rhodium.
  • g.
    Add 150 μL of 2% FA with Rhodium and mix by pipetting.

    Note: DNA staining is indispensable for CyTOF to identify single cell events. Iridium is a common alternative but is avoided here due to the potential spillover signal to 194Pt.

  • h.
    Wrap the sample tube in aluminum foil and put it in the fridge (4°C) overnight or up to 48 h.

    Note: Storing stained cells in FA may be done up to 14 days, while it is preferable to acquire data within 48 h.

    Note: Once opened, 16% FA can be used for up to one week.

Table 2.

Antibody cocktails for each CyTOF staining step

Label	Target	Clone	Conjugation using Maxpar Labeling Kit (y/n)	Dilution	Volume (μL)
Chemokine receptor marker stain
127I	IDU	–	N	1:1000	0.1
141Pr	CCR6 (CD196)	G034E3	N	1:100	1
156Gd	CXCR3 (CD183)	G025H7	N	1:100	1
167Er	CCR7 (CD197)	G043H7	N	1:100	1
168Er	CCR5 (CD195)	J418F1	Y	1:50	2
171Yb	CXCR5 (CD185)	RF8B2	N	1:100	1
175Lu	CCR4 (CD194)	L291H4	N	1:50	2
Buffer: cRPMI					41.9
Volume of chemokine receptor marker stain					50
Total volume when added to the sample					100
Cell surface marker stain
89Y	CD45	HI30	n	1:100	1
111Cd	TCRαβ	IP26	y	1:25	4
112Cd	CD33	WM53	y	1:100	1
113In	CD14	M5E2	y	1:25	4
115In	FMC63/Biotin	Y45/1D4-C5	y	1:25	4
139La	CD39	A1	y	1:12.5	8
142Nd	CD19	HIB19	n	1:100	1
143Nd	HLA-DR	L243	n	1:200	0.5
144Nd	CD38	HIT2	n	1:100	1
145Nd	CD4	RPA-T4	n	1:100	1
146Nd	CD8a	RPA-T8	n	1:50	2
147Sm	ICOS	C398.4A	y	1:100	1
148Nd	PD-L1 (CD274)	29E.2A3	n	1:100	1
149Sm	CD25	2A3	n	1:100	1
150Nd	OX40 (CD134)	ACT35	n	1:25	4
151Eu	CD57	HNK-1	y	1:400	0.25
152Sm	TCRγδ	11F2	n	1:25	4
154Sm	CD3	UCHT1	n	1:100	1
155Gd	CD27	L128	n	1:200	0.5
158Gd	CD10	HI10a	n	1:100	1
160Gd	CD73	AD2	y	1:50	2
161Dy	CD22	HIB22	y	1:100	1
163Dy	CD56	NCAM16.2	n	1:200	0.5
164Dy	CD161	HP-3G10	n	1:100	1
165Ho	Vα24	6B11	y	1:12.5	8
166Er	CD34	581	n	1:100	1
169Tm	CD16	3G8	y	1:100	1
170Er	CD45RA	HI100	n	1:400	0.25
172Yb	Tim-3	F38-2E2	y	1:50	2
173Yb	TIGIT	A15153G	y	1:25	4
174Yb	PD-1 (CD279)	EH12.2H7	n	1:50	2
176Yb	CD127	A019D5	n	1:200	0.5
209Bi	CD11b	ICRF44	n	1:200	0.5
Buffer: CSM					5
Volume of cell surface marker stain					70
Total volume when added to the sample					100
Intracellular maker stain
159Tb	CTLA-4 (CD152)	L3D10	y	1:100	1
162Dy	FoxP3	236A/E7	y	1:25	4
Buffer: 1× perm buffer					45
Volume of intracellular marker stain					50
Total volume when added to the sample					100

CyTOF data acquisition

Timing: 3–6 h

When acquiring data, it is important to handle samples carefully so as not to lose cellular events.

• 7.
  CyTOF setup.

  • a.
    Turn on Helios™ mass cytometer (or CyTOF XT™) and perform daily setup, tuning, and bead quality control according to manufacturer’s instructions (https://standardbio.com/support/instrument-support/cytof-xt-system-support/#documents).
• 8.
  Sample preparation before data acquisition.

  • a.
    Wash sample once with 2 mL CSM followed by twice with 2 mL Maxpar® Cell Acquisition Solution (CAS) by centrifugation at 800× g for 5 min, 4°C. Count cells prior to centrifugation in final wash.
  • b.
    After final spin, remove the supernatant by careful aspiration using a pipette and leave cells in pellet on ice.
  • c.
    Immediately before running, add CAS +15% EQ Four Element Calibration Beads, to give 1 million cells/mL. Filter sample using Falcon® Cell Strainer tube (35 μm) immediately before run. Troubleshooting 2.
• 9.
  Data acquisition.

  • a.
    Collect sample data at 200–500 events per second. Cell events higher than this may result in sample line clogging. Troubleshooting 3.

Manual gate-based barcode removal and data analysis

Timing: 1 h or more

After normalizing the data, single-cell data is extracted and barcode removal is performed to obtain data for each sample. Data analysis can be carried out in a variety of ways depending on the needs of a researcher.

• 10.
  Normalization and Concatenation of the acquired data.

  • a.
    Normalize FCS files to EQ bead signal with CyTOF Software provided by Standard BioTools according to manufacturer’s instructions (https://standardbio.com/support/instrument-support/cytof-xt-system-support/#documents). If you measure one sample in several parts, concatenate the data after normalization.

Note: FCS (Flow Cytometry Standard) is a file format developed by the International Society for Advancement of Cytometry (ISAC) to store data generated by flow and mass cytometers.

• 11.
  Data cleanup and barcode removal.

  • a.
    Gating and manual B2MG+CD147 barcode removal can be performed in Cytobank software or alternatives such as FlowJo™. Gating strategy for data cleanup and barcode removal (serial gating of 195Pt/196Pt, 194Pt/198Pt, and 114Cd/116Cd) is shown in Figure 1. In the barcode removal, the plot area is divided diagonally into two parts, and a contour plot is used to illustrate the area containing 95% of the cell population in each region. Gating is then performed along this 95% line to extract the cell population. This process is repeated to identify the original samples. Troubleshooting 4.

Note: In serial gating, where cell populations are arranged diagonally as shown in Figure 1, double positive fractions are considered doublets. Generally, the proportion of doublet cells should be less than 10% of the total. Also, if double negative fractions appear, it is possible that the barcode antibodies have not stained samples properly.

Note: If there are no particular problems with staining, barcode removal using CATALYST is comparable with the manual barcode removal.¹⁰ If you would like to further expand the scale of barcode samples, you could use CATALYST. However, since distribution of the cell population in bone marrow samples tends to be wide, be sure to check that the separation is successful. The following is an example of CATALYST code for barcode removal in R. The sample data before and after barcode removal is deposited in Zenodo (https://zenodo.org/) under accession number 20046563.

# load packages

library(flowCore)

library(CATALYST)

library(SingleCellExperiment)

# FCS loading

fcs_file <- "relapse_i_samples_after_Gaussian_filter.fcs"

ff <- read.FCS(fcs_file, transformation = FALSE, truncate_max_range = FALSE)

# channel check

colnames(ff)

pData(parameters(ff))[, c("name", "desc")]

# panel creation(all marker_class is "type")

panel <- data.frame(fcs_colname = colnames(ff), antigen = pData(parameters(ff))$desc, marker_class = "type", stringsAsFactors = FALSE)

# desc complement

panel$antigen[is.na(panel$antigen) | panel$antigen == ""] <-

 panel$fcs_colname[is.na(panel$antigen) | panel$antigen == ""]

# prepData

sce <- prepData(ff, panel = panel)

# bc_key creation(Column name is mass number)

bc_key <- data.frame(

 "114" = c(0, 1, 0, 1, 0, 1, 0, 1),

 "116" = c(1, 0, 1, 0, 1, 0, 1, 0),

 "194" = c(0, 0, 1, 1, 0, 0, 1, 1),

 "195" = c(0, 0, 0, 0, 1, 1, 1, 1),

 "196" = c(1, 1, 1, 1, 0, 0, 0, 0),

 "198" = c(1, 1, 0, 0, 1, 1, 0, 0),

 check.names = FALSE)

rownames(bc_key) <- paste0("S", 1:8)

# barcode removal

sce <- assignPrelim(sce, bc_key)

sce <- estCutoffs(sce)

sce <- applyCutoffs(sce)

# confirm results

table(sce$bc_id)

prop.table(table(sce$bc_id))

# QC plot

plotYields(sce)

plotEvents(sce)

# Mahalanobis plot

for (s in setdiff(unique(sce$bc_id), "0")) {print(plotMahal(sce, which = s))}

# export fcs files for each sample

out_dir <- "debarcoded_fcs"

dir.create(out_dir, showWarnings = FALSE)

for (s in setdiff(unique(sce$bc_id), "0")) {

 sce_sub <- sce[, sce$bc_id == s]

 ff_sub <- sce2fcs(sce_sub)

 write.FCS(ff_sub, filename = file.path(out_dir, paste0(s, ".fcs")))}

• 12.
  Identifying cell populations and comparing their frequencies

  • a.
    The subsequent analysis involves identifying cell populations and comparing their frequencies. While this analysis can be performed using conventional two-axis gating (Figure 2), dimensionality reduction and clustering enable a more detailed and comprehensive analysis.

Figure 1.

Gating strategy performed in Cytobank software for data cleanup and barcode removal

After EQ beads removal, cell events are recognized by DNA staining (rhodium in this protocol). Doublets are removed using event length parameter and dead cells by positivity for palladium. Since palladium reagents contain nearly equal amounts of 106Pd and 108Pd isotopes, the distribution is similar in both the 106Pd and 108Pd channels.

Gaussian parameters (center shown here but residual offset and length can also be used) are used for selecting high quality signal pulses. Sequential gating of barcode removal separates cell populations diagonally and identifies the original samples. In the barcode removal gating, the population that appears in the double positive area is considered remaining doublets.

Figure 2.

Gating of major immune cell populations and T cell subsets stained with this protocol

Two-dimensional gating of the CyTOF data after data cleanup shows major immune cell populations and T cell subsets. This type of analysis can be performed not only with Cytobank but also with FlowJo™, similar to analyzing flow cytometry data.

Expected outcomes

This protocol enables efficient acquisition of high-parameter single-cell data from multiple human hematological malignancy samples. The amount of single-cell data obtained compared to the initial number of input cells depends on the sample type and processing steps. Generally, CyTOF acquisition of PBMCs leads to higher yield, while the recovery rates for tissue-derived samples such as bone marrow aspirates are lower. When using this protocol with cryopreserved human hematological malignancy samples, the ratio of measured events to the number of cells at the start of staining are approximately 30%–40%. Therefore, the number of cells used for staining should be determined based on the proportion of the cell population of interest and the expected cell loss during the staining and data acquisition.

Live cell barcoding enables analysis of serial samples obtained from the same patient under more physiological conditions than barcoding which requires cell fixation, thereby minimizing experimental batch effects. Such serial sample data can be analyzed collectively through dimensionality reduction, clustering, cell population annotation, and comparison of each population, as demonstrated in Mikami et al.^1,11 Investigating how cancer cells and immune cell populations change with treatment and disease progression is essential for understanding the pathology.

Limitations

This protocol has been optimized for the analysis of human hematological malignancy samples using CyTOF and requires prior validation before application to other tumor samples, particularly to confirm whether B2MG- and CD147-based barcoding is applicable. It should also be noted that cryopreservation of specimens prior to CyTOF staining results in the loss of most myeloid and dendritic cells from the immune cell fraction. When analyzing these cell populations, cells should be collected from fresh specimens using appropriate procedures.

Another key limitation of this protocol is that functional markers such as phospho-proteins and cytokines must be evaluated under appropriate conditions. For example, to assess phospho-proteins, cells should be fixed immediately after epitope activation to preserve their phosphorylation state. In addition, when measuring cytokines, it is sometimes necessary to add protein secretion inhibitors such as brefeldin or monensin several hours before cell staining. In such cases, consider using Pd-based barcoding which involves fixation first, or modify the staining protocol accordingly.

Troubleshooting

Problem 1

Thawing samples that are expected to contain a high number of dead cells.

Potential solution

Frozen samples of mononuclear cells obtained from bone marrow or tissues tend to contain a large number of dead cells. In such cases, adding DNase to cRPMI can reduce cell clumping due to eluted DNA. In such cases, using cRPMI+DNAse (adding 4 μL of Pierce™ Universal Nuclease stock to 50 mL of cRPMI, final concentration: 1000 U/50 mL cRPMI) at the first dilution upon thawing can reduce cell clumping.

Problem 2

Although there has been a substantial number of cells at the time of staining, the number of cell events measured by CyTOF is low.

Potential solution

It is preferable to keep samples pelleted if you leave them for a long time. When multiple samples are run, the next sample should be resuspended and prepared while the previous sample is being acquired.

Problem 3

The sample line frequently becomes clogged during measurements.

Potential solution

Remove the sample, refilter, and dilute sufficiently to obtain a flow rate of 100 events per second or less. However, diluted cell suspensions take a longer time to acquire data.

Problem 4

Serial gating does not clearly separate samples.

Potential solution

There may be cases where the samples are not separated clearly due to insufficient barcode staining. Originally, we suggest the serial gating in the order of 195Pt/196Pt, 194Pt/198Pt, and 114Cd/116Cd. However, the separation of cell population could be improved by using the combinations for gating in order starting from the better staining combination in each case (e.g., 114Cd/116Cd, 195Pt/196Pt, and then 194Pt/198Pt). If separation is still difficult, use a contour plot to illustrate the boundary between the two cell populations and perform gating along that line, although some mixing of sample data is unavoidable.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Itaru Kato (itarkt@kuhp.kyoto-u.ac.jp).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Takashi Mikami (mikami.takashi.3r@kyoto-u.ac.jp).

Materials availability

This study did not generate unique reagents or materials.

Data and code availability

The original CyTOF data have been deposited to Zenodo (https://zenodo.org/) under accession nos. Zenodo: 13731333, 13731405, and 20046564. This study did not report original code.

Acknowledgments

This study was supported by the Japan Society of Promotion of Science (JSPS) KAKENHI (nos. JP20H00528, JP21K19405, JP23K18264, and JP24H00628 to J.T.; and no. JP22K07211 to I.K.); by the Project for Promotion of Cancer Research and Therapeutic Evolution (P-PROMOTE) from AMED (nos. JP22cm0106xxxh000x, 23ama221505h0002, 24ama221531h0001, and 25ama221531h0002 to J.T.; and nos. 22ama221514h0001 and 23ama221514h002 to I.K.); by the Practical Research for Innovative Cancer Control from AMED (no. JP21ck0106531 to I.K.); by the Princess Takamatsu Cancer Research Fund to J.T.; by the Takeda Hosho Grants for Research in Medicine to J.T.; by the internal grant Ishizue from Kyoto University Research Administration to J.T.; by the Mother and Child Health Foundation to I.K.; and by the Japan Leukemia Research Fund to I.K. The graphical abstract was created in BioRender (Immune, R. (2026) https://BioRender.com/oj9hsn1) and Adobe Illustrator 2025. The authors would like to thank Rika Ishii and Miho Itoh-Takahashi for technical support.

Author contributions

T.M. and I.K. designed the research. J.B.W. provided guidance and advice on CyTOF staining methods. T.M. performed the experiments and analyzed the data. I.K., J.B.W., and J.T. interpreted the data and contributed to discussion. T.M. wrote the manuscript.

Declaration of interests

The authors declare no competing interests.