Cell-based ELISpot protocol to detect and quantify antigen-specific antibody-secreting cells in murine whole-organ single-cell suspensions

Summary

Antibody-secreting cells (ASCs) are the hallmark of the adaptive immune response to vaccines or infections. Here, we present a protocol to quantify the number of ASCs using an enzyme-linked immunospot assay (ELISpot). We describe the preparation of cell samples, ELISpot plates, and their quantification. This assay has potential applications in vaccine studies and research on the adaptive immune system or ASC-associated diseases.

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

Graphical abstract

Highlights

• •Instructions for the preparation of murine single-cell suspensions
• •Steps for seeding cell suspensions onto ELISpot plates for high-throughput analysis
• •Guide to processing ELISpot plates with additional tips to improve spot quality
• •Detailed spot quantification instructions

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

Antibody-secreting cells (ASCs) are the hallmark of the adaptive immune response to vaccines or infections. Here, we present a protocol to quantify the number of ASCs using an enzyme-linked immunospot assay (ELISpot). We describe the preparation of cell samples, ELISpot plates, and their quantification. This assay has potential applications in vaccine studies and research on the adaptive immune system or ASC-associated diseases.

Before you begin

The differentiation of activated B cells into antibody-secreting cells (ASCs) is the basis for the humoral immune response of the adaptive immune system to an antigen acquired by infection or vaccination.² When naive B cells are activated by binding to an antigen with their specific B cell receptor (Immunoglobulin M or IgM), they can induce a germinal center reaction with the help of T cells that have specificity against the same antigen.³ During the germinal center formation, the antigen specificity of the B cell receptor can be increased and the isotype of its Ig heavy (H) chain can switch from μH (IgM) to αH (IgA), γH (IgG) or εH (IgE). After these B cells differentiate into ASCs, they secrete the soluble version of the fine-tuned B cell receptor, resulting in highly antigen-affine antibodies with different functions depending on their IgH-chain isotype. A subpopulation of ASCs can mature into long-lived plasma cells that survive for decades in specialized niches in the bone marrow or intestine, continuously secreting antibodies and protecting against recurrent pathogens.^2,4,5 However, pathogenic ASCs can induce autoimmune diseases or degenerate into multiple myeloma cells. Therefore, we established and refined the protocol described here to detect ASCs of all IgH chains based on their secretory function and the antigen specificity of their secreted antibodies. This protocol can be used for basic studies of the adaptive immune response, vaccine studies or studies analyzing antibody-associated diseases or pathogenic ASCs. Institutional and governmental approval must be obtained to perform the described assay using primary mouse cells, especially from immunized animals, and institutional and national guidelines and regulations for animal experimentation must be strictly followed.

Institutional permissions

Mice were housed under pathogen-free conditions in accordance with national and institutional guidelines at the Preclinical Experimental Animal Center (PETZ) or the Nikolaus-Fiebiger Center (NFZ) of the University of Erlangen-Nürnberg, Erlangen, Germany. All animal experiments were performed in accordance with institutional and national guidelines and were approved by the State of Bavaria (Amt für Veterinärwesen und gesundheitlichen Verbraucherschutz der Stadt Erlangen, Erlangen, Germany; Regierung von Unterfranken, Würzburg, Germany).

Preparation of ELISpot plates

Timing: ∼0.5 h per plate, 1 h and 12–24 h incubation

In this step, the ELISpot plates are coated with the capture antibody or the antigen of interest (here (4-hydroxy-3-nitrophenyl) acetyl [NP]) and prepared for incubation with the cell suspension.

CRITICAL: Preparation of buffers and solutions is detailed in the chapter “Materials and equipment setup”. All reagents and buffers should be handled and disposed of in accordance with institutional and national guidelines and safety regulations.

• 1.
  Coat the 96-well plates with the capture antibody or the antigen.

  • a.
    Dilute the capture antibody or the antigen in ELISpot coating buffer.

    • i.
      To analyze total Ig-secreting cells, prepare a goat anti-mouse IgM, IgG or IgA- solution at 2 μg/mL.
    • ii.
      To analyze antigen-specific Ig-secreting cells, prepare a 1 μg/mL solution of NP-BSA.

      Note: Under normal circumstances, using medium adsorption 96-well flat bottom plates is sufficient. However, depending on the antigen you are using, the maxisorp 96-well flat bottom plates are recommended for more intense spots and reliable results.

  • b.
    Transfer the antibody or antigen solution into the 96-well plates using a multichannel pipette.

    • i.
      To detect total Ig-secreting cells, add 50 μL of the prepared antibody solution to each well.
    • ii.
      To detect antigen-specific Ig-secreting cells add 100 μL of the prepared antigen-solution to each well.

      CRITICAL: It may be necessary to determine the best working concentration for your individual antigen.

  • c.
    Carefully and gently tap the sides of the plates with your hand until the entire bottom of each well is evenly covered with the solution.
  • d.
    Stack the plates, cover the top plate with a fitting lid, and transfer the stack into a plastic bag to prevent evaporation.
  • e.
    Incubate the plates ∼12–24 h at 4°C.

    Note: To speed up all pipetting steps in this protocol, we recommend using a multichannel pipette.

• 2.
  Wash the plates.

  • a.
    Discard the coating buffer in one swift motion by inverting the plate.
  • b.
    Immerse the plate in a bucket of washing buffer to fill all wells.
  • c.
    Discard the washing buffer in one swift motion by inverting the plate.
  • d.
    Repeat Steps b and c for a total of three times.
  • e.
    To remove most of the washing buffer, tap the plates firmly upside down on a stack of paper towels.

Note: Alternatively, the multichannel pipette can be used to add 200 μL of washing buffer per well for washing.

CRITICAL: To analyze total Ig-secreting cells, wash the plates with 1x PBS. To analyze antigen specific Ig-secreting cells, wash the plates with 1x PBS – 0.05% Tween.

CRITICAL: Do not allow the plates to dry completely from this step. Once the contents of the plates have been discarded and tapped dry, always work quickly when loading the wells with the new solution.

• 3.
  Block uncoated areas on the plates thereby preventing unspecific binding of secreted antibodies to the wells.

  • a.
    Using a multichannel pipette add 200 μL of blocking buffer to each well.
  • b.
    Stack the plates, cover the top plate with an appropriate lid, and transfer the stack in a plastic bag to prevent evaporation.
  • c.
    Incubate the plates for 1 h at 37°C.

CRITICAL: To analyze total Ig-secreting cells, block the plates with 1% gelatin in 1x PBS. To analyze antigen specific Ig-secreting cells, block the plates with 1% Bovine Serum Albumin (BSA) in 1x PBS.

• 4.
  Wash the plates.

  • a.
    Discard blocking buffer in one swift motion by inverting the plate.
  • b.
    Immerse the plate in a bucket of washing buffer to fill all wells.

    CRITICAL: To analyze total Ig-secreting cells, wash the plates with 1x PBS. To analyze antigen specific Ig-secreting cells, wash the plates with 1x PBS – 0.05% Tween.

  • c.
    Discard the washing buffer in one swift motion by inverting the plate.
  • d.
    Repeat Steps b and c for a total of three times.
  • e.
    To remove most of the washing buffer, tap the plates firmly upside down on a stack of paper towel.

    CRITICAL: After the last washing step with 1x PBS – 0.05% Tween, wash one more time with 1x PBS before incubating the cells in the plates in Step 3 of "step-by-step method details." Since Tween is a detergent that solubilizes the cell membrane, it must be completely removed.

    CRITICAL: Avoid completely drying out the plates. Therefore, do not leave the plates without buffer between steps. Remove the washing buffer immediately before proceeding to Step 2 of "step-by-step method details.

    Troubleshooting 8.

Preparation of murine single-cell suspensions

Timing: ∼45 min

This step describes the preparation of a single cell suspension from murine spleen. To prepare cell suspensions from other organs (mouse or human), the protocol must be adapted. Of course, single cell suspensions prepared from cell cultures or cell lines using an appropriate protocol can also be analyzed. To detect antigen-specific ASCs, mice must be immunized using an immunization scheme that induces the formation of ASCs with the IgH-chain isotype of interest. This protocol has already been successfully used for a variety of vaccines and immunization strategies.^1,6,7,8

• 5.
  Isolate the murine spleen.

  • a.
    Euthanize the mouse according to institutional and national guidelines.
  • b.
    Sterilize the outside of the mouse with 70% ethanol.
  • c.
    Carefully open the abdomen of the mouse and retrieve the spleen.
  • d.
    Remove any attached tissue, such as fat, from the spleen.
  • e.
    Transfer the spleen to a 70 μm cell strainer placed in a petri dish containing 1 mL of 1x PBS-1% FCS buffer.

Note: Single cell suspensions can be isolated from any organ of interest. Alternatively, single cell suspensions from cell lines or activated primary cells may be used.

• 6.
  Homogenize the spleen.

  • a.
    Using clean scissors, gently mince the spleen in the 70 μm cell strainer.
  • b.
    Use the end of a 2 mL syringe plunger to gently mash the spleen through the cell strainer.
  • c.
    After mashing, wash the remainder of the spleen on the syringe plunger through the 70 μm cell strainer into the petri dish by rinsing with 1 mL of 1x PBS-1% FCS buffer.
  • d.
    Wash the remainder of the spleen single cells from the 70 μm cell strainer into the petri dish by gently rinsing it with 1 mL of 1x PBS-1% FCS buffer while holding it slightly above the petri dish.
  • e.
    While still holding the 70 μm cell strainer above the petri dish, recover the remaining suspension from the underside of the 70 μm cell strainer by shifting it slightly and aspirating it with a 1 mL micropipette.
  • f.
    Add the recovered solution to the contents of the petri dish.
  • g.
    Transfer the single cell suspension from the petri dish to a 15 mL reaction tube.
  • h.
    Centrifuge the tube at 470 g and 4°C for 7 min.
  • i.
    Discard the supernatant in one swift motion by inverting the tube.

Note: Mincing the spleen prior to homogenization makes the process gentler and increases cell viability. Do not use excessive force when mashing the tissue.

• 7.
  Lyse the erythrocytes in the splenic single cell suspensions.

  • a.
    Heat the 1x red blood cell lysis buffer to ∼20°C–22°C (or max. 37°C).
  • b.
    Suspend the cell pellet in 6 mL of warm red blood cell lysis buffer.
  • c.
    Incubate the cells for 6 min at ∼20°C–22°C.
  • d.
    Stop the reaction by adding 6 mL of 1x PBS-1% FCS buffer to the sample and gently mix by pipetting up and down or by inverting the tube after closing the lid.

CRITICAL: It is important to strictly stick to the incubation time. Prolonged incubation with red blood cell lysis buffer will also destroy the lymphocytes.

Note: When incubating more than one sample, first add the red blood cell lysis buffer to each cell pellet without resuspending it. Then begin the process by resuspending the first sample and starting your timer. Each resuspension step may take you 20–30 s, so always check the elapsed time when starting to resuspend the next sample. This is important for proper timing when stopping the reaction. Stop each reaction after exactly 6 min.

Note: When analyzing lymphatic organs that are not supplied with blood or even cell lines, this step can be skipped.

• 8.
  Finalize the single cell suspension.

  • a.
    To remove cell debris, filter the cell suspension through a 30 μm cell filter into a fresh 15 mL reaction tube.
  • b.
    Rinse the remaining cells from the filter into the tube with 1 mL of 1x PBS-1% FCS buffer.
  • c.
    Centrifuge the tube at 470g and 4°C for 7 min.
  • d.
    Discard the supernatant in one swift motion by inverting the tube.
  • e.
    Resuspend the cells in complete medium to a final volume of 500 μL.
  • f.
    Count the cells.

CRITICAL: It is important to resuspend the cells in a small volume to avoid additional centrifugation in the following steps.

Troubleshooting 1.

Troubleshooting 7.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Goat anti-mouse IgA, alkaline phosphatase (AP) (working dilution 1:4,000)	SouthernBiotech	RRID: AB_2794372
Goat anti-mouse IgA, unconjugated (working concentration 2 μg/mL)	SouthernBiotech	RRID: AB_2314669
Goat anti-mouse IgG, alkaline phosphatase (AP) (working dilution 1:4,000)	SouthernBiotech	RRID: AB_2794293
Goat anti-mouse IgG, unconjugated (working concentration 2 μg/mL)	SouthernBiotech	RRID: AB_2794290
Goat anti-mouse IgM, alkaline phosphatase (AP) (Working dilution 1:4,000)	SouthernBiotech	RRID: AB_2794200
Goat anti-mouse IgM, unconjugated (working concentration 2 μg/mL)	SouthernBiotech	RRID: AB_2794197
Chemicals, peptides, and recombinant proteins
1.5 M AMP 100 mL, pH 10.3 (2-amino-2-methyl-1-propanol) 90% tech.grade → dilute stock for 1.5 M	Sigma/Merck	Cat# A65182
BCIP	Sigma/Merck	Cat# B8503
Beta-mercaptoethanol 50 mM	Thermo Fisher Scientific (Gibco)	Ref. 31350-010
BSA (bovine serum albumin, albumin fraction 5.98%)	Carl Roth	Cat# 8076.4
DMSO	Roth	Cat# 4720.1
Fetal bovine (calf) serum (FBS/FCS)	Thermo Fisher Scientific (Gibco)	Ref.: A5256701
Gelatin	Merck Millipore	Cat# 104078
KCl	Roth	Cat# 6781.1
KH2Po4	Roth	Cat# 3904.1
L-glutamine 200 mM	Thermo Fisher Scientific (Gibco)	Ref. 25030-024
1 M MgCl2	N/A	N/A
NaCl	Roth	Cat# 0962.2
15 mM Na2CO3	Sigma/Merck	Cat# 1.06392
35 mM NaHCO3	Sigma/Merck	Cat# 1.06329
Na2HPo4-7H2O or Na2HPO4	Roth	Cat# P030.3
NaN3 10%	N/A	N/A
NP-BSA	Biosearch Technologies	Cat# N-5060-25
Penicillin-Streptomycin (10,000 U/mL)	Thermo Fisher Scientific (Gibco)	Ref. 15140-122
RBC Lysis Buffer (10X)	BioLegend	Cat# 420302
RPMI 1640	Thermo Fisher Scientific (Gibco)	Ref. 31870
Sodium pyruvate 100 mM	Thermo Fisher Scientific (Gibco)	Ref. 11360-039
Tween 20	Sigma/Merck	Cat# P1379
Triton X-405	Sigma/Merck	Cat# T-7253
Experimental models: Organism/strains
Mus musculus C57BL/6	In-house breeding; female or male, 18–47 weeks	N/A
Mus musculus RAG2-KO	Generated by Shinkai et al.,9 in-house breeding, male, 19 weeks	N/A
Software and algorithms
BioSpotR ImmunoSpot 5.1.36	C.T.L.	RRID: SCR_011082
Others
Flat bottomed Maxisorp 96-well ELISA plates	Thermo Fisher Scientific	Cat# 442404
Flat bottomed conventional 96-well ELISA plates	Greiner	Cat# 655001
Nalgene Rapid-Flow Disposable sterile filtration equipment	Thermo Fisher Scientific	Cat# 568-0020
ImmunoSpotR Series 6 Ultra-V Analyzer	C.T.L.	N/A

Materials and equipment

10x PBS-Stock pH 7.4

Reagent	Final concentration	Amount
NaCl	1.369 M	320 g
KCl	0.027 M	8 g
Na2HPO4- 7 H2O or Na2HPO4	0.010 M	57.6 g 46 g
KH2PO4	0.018 M	9.6 g
MiliQ-H2O	N/A	Add up to a total of 4 L
Total	N/A	4 L

Store at ∼20–22 °C. Without contamination, there is no precise expiration time.

Substrate buffer/AMP 10x buffer, pH value = 10.25

Reagent	Final concentration	Amount
1.5 M AMP, pH: 10.3	1 M	100 mL
1 M MgCl2	0.005 M	750 μL
Triton X-405	N/A	152 μL
10% NaN3	0.1%	1.5 mL
MiliQ-H2O	N/A	Add up for a final amount of 150 mL stepwise. Adjust pH before adding the final volume.
Total	N/A	150 mL

Store at 4°C protected from light. Without contamination or changes in color, there is no precise expiration time.

Complete medium (R10)

Reagent	Final concentration	Amount
RPMI 1640	N/A	500 mL
FCS (heat inactivated)	10%	50 mL
L-Glutamine 200 mM	4 mM	10 mL
Penicillin-Streptomycin (10,000 U/mL)	100,000 U	5 mL
Sodium Pyruvate 100 mM	1 mM	5 mL
Beta-mercaptoethanol 50 mM	0.05 mM	0.5 mL
Total	N/A	570.5 mL

Store at 4°C. Without contamination or obvious changes in pH the expiration time of the ingredients should be considered.

ESA substrate solution stock

• •Substrate buffer (AMP 10x buffer), 50 mL.
• •BCIP 500 mg.
• •In 450 mL distilled water.
• •Stir at ∼20°C–22°C until no solid remains.

(Store at 4°C protected from light. Without contamination or changes in color, there is no precise expiration time.)

Ready to use ESA substrate working solution

• •For one 96-well plate, mix 4 mL ESA substrate solution stock with 1.5 mL of distilled water.

(Store ESA substrate working solution at 4°C, protected from light, and prepare directly before use.)

Coating buffer

• •35 mM NaHCO₃, 1.47 g.
• •15 mM Na₂CO₃, 795 mg.
• •In 500 mL distilled water.

(Store at 4°C. Without contamination, there is no precise expiration time.)

1x PBS (5 L)

• •500 mL 10x PBS.
• •4.5 L MiliQ-H₂O.

(Store at ∼20°C–22°C. Without contamination, there is no precise expiration time.)

Washing buffer: 1x PBS + 0.05% Tween 20 (2 L)

• •2 L 1x PBS.
• •1 mL Tween 20.

(Store at ∼20°C–22°C. Without contamination, there is no precise expiration time.)

Washing buffer: Water +0.1% Tween 20 (1 L)

• •1 L distilled water.
• •1 mL of Tween 20.

(Store at ∼20°C–22°C. Without contamination, there is no precise expiration time.)

Blocking buffer: 1x PBS + 1% Bovine serum albumin (BSA)

• •1 g BSA.
• •100 mL of 1x PBS.
• •shake or stir at ∼20°C–22°C until no solid BSA can be seen.

(Store at 4°C. Do not store longer than 48 h.)

Blocking buffer: 1x PBS + 1% gelatin

• •1 g Gelatin.
• •100 mL of 1x PBS.
• •Carefully boil in the microwave or water bath until no solid gelatin remains.
• •Temperature before usage should be < 37°C.

(Store at 4°C. Do not store longer than 48 h.)

1x PBS + 1% gelatin + 1% Tween 20

• •100 mL 1x PBS.
• •1 g gelatin.
• •Carefully boil in the microwave or water bath until no solid gelatin remains.

(Can be prepared from Gelatin blocking buffer, reheated to 37°C in a water bath.)

• •1 mL Tween 20.

Do not store the buffer for longer periods of time. Prepare it fresh for each experiment.

CRITICAL: Gelatin- when boiling the gelatin, pay attention to evaporation and the risk of scalding. Substrate Buffer- when using AMP, pay attention to the color of the solution. If a color change to blue can be seen, the buffer should be discarded and prepared freshly.

CRITICAL: If not already prepared under sterile conditions, all buffers should be sterilely filtered before longer storage periods.

Step-by-step method details

Seeding and incubation of single-cell solutions

Timing: ∼0.5 h per plate preparation, 12–24 h incubation

This step describes the seeding of the already prepared single cell suspension (Steps 5–8 of “before you begin”) into the also prepared ELISpot plates (Steps 1–4 of “before you begin”). The instructions include a detailed description of the preparation of the cell dilution series.

• 1.
  Prepare the single cell suspension to the desired concentrations.

  • a.
    Calculate the volume for the required number of cells for each sample, including an excess.

    Note: For example, to detect total IgG using technical triplicates, you will need 450000 cells (150000 cells/well) per sample. Calculate the volume required for 500000 cells per sample.

  • b.
    Transfer the required volume for the desired number of cells from each sample to fresh tubes.
  • c.
    Add complete medium to the cell solutions in the fresh tubes to achieve the desired cell concentration with a total volume of 150 μL per well and gently resuspend the cells.

    Note: For example, to detect total IgG, a concentration of 1 × 10⁶ cells/mL (150000 cells in 150 μL) is required. Including excess, transfer 500000 cells per sample to a fresh tube (see Steps 1a and 1b of “step-by-step method details”). Then, add enough complete medium to reach a final volume of 500 μL. For correct cell concentration, DO NOT simply add 500 μL to your cell solution. Consider the volume that is already in the tube.

    CRITICAL: To analyze antigen-specific Ig-secreting cells, resuspend the cells in complete medium-1% BSA.

• 2.
  Prepare the coated plates for seeding the cells in a dilution series.

  • a.
    Add 100 μL of complete medium to all wells of the plates prepared in Steps 1–4 of “before you begin” except for the first row (Figure 1A).

CRITICAL: To analyze antigen-specific Ig-secreting cells, add 100 μL of complete medium-1% BSA to all wells except the first row. BSA is not required but will reduce the background.

CRITICAL: Do not allow plates to dry completely between steps. Discard the washing buffer in Step 4 of “before you begin” immediately before proceeding with this step.

• 3.
  Seed the prepared cell solutions in a serial dilution (Figure 1A).

  • a.
    Add 150 μL of the resuspended cell suspensions at the desired concentrations (Step 1 of “step-by-step method details”) to each well of the first row of the prepared plates (Step 2 of “step-by-step method details”) according to your plate layout.
  • b.
    Using a multichannel pipette, transfer 50 μL from each well of the first row to the corresponding well of the second row, which already contains 100 μL of complete medium.

    Note: The first row now contains 100000 cells in 100 μL volume per well.

  • c.
    Carefully resuspend the cells in the wells of the second row in the now total volume of 150 μL by pipetting up- and down very slowly at least 5 times.
  • d.
    Using a multichannel pipette, transfer 50 μL from each well of the second row to the corresponding well of the third row, which already contains 100 μL of complete medium.

    Note: The second row now contains approximately 33000 cells in 100 μL of complete medium.

  • e.
    Carefully resuspend the cells in the wells of the third row in the now total volume of 150 μL by pipetting up- and down very slowly at least 5 times.
  • f.
    Repeat Steps 3d and 3e of “step-by-step method details” for each row until you reach the last row of the plate.
  • g.
    After carefully resuspending the cells in the last row, discard 50 μL of this cell suspension.

    Note: This leaves a final volume of 100 μL in each row of the plate.

    CRITICAL: In these steps, it is important to always resuspend the cells well, but very carefully to ensure correct dilution of the samples, but to avoid shear stress and therefore cell damage that could bias the final results.

    CRITICAL: Avoid scratching the bottom of the wells with the pipette tip as this will remove the coating and alter your results.

    Note: It is helpful, but not required, to use a multichannel pipette for homogeneous results.

• 4.
  Incubate the cell suspensions.

  • a.
    Cover the plates with appropriate lids to prevent evaporation.
  • b.
    Check the plates under the microscope for proper seeding.
  • c.
    Incubate the plates at 37°C with 5% of CO₂ for ∼12–24 h.

Note: From now on, the protocols for total Ig secreting cells and antigen-specific Ig secreting cells are the same.

Figure 1.

Plate scheme and dilution steps for a 96-well plate used in an ELISpot assay

(A) Schematic depiction of a 96-well plate prepared for an ELISpot assay. Major points 1–3 are performed in the indicated order (Described in Step 3 of “step-by-step method details”). Major point 3 “Pipette serial dilution” includes 15 sub-points, performed in the numbered order.

(B) Step-by-step description of how to calculate ASCs using a spreadsheet. In the example, 100000 splenocytes were seeded per well in the first row to analyze the number of total IgM-secreting cells in the spleen. A 1:3 dilution was used in the following rows. For final quantification, the spreadsheet generated after quality control was used for reliable spot counts per well (Step 15 of “step-by-step method details”). “-2″ is the code for a well that could not be used for quantification because too many spots in the well made it impossible to separate them from each other (Step 14e of “step-by-step method details”).

Troubleshooting 6.

Processing of ELISpot plates

Timing: ∼0.5 h per plate preparation, 2 h and 12–24 h incubation

This step describes the processing of the ELISpot plates after incubation with the single cell suspensions. This includes the finalization of the plates by developing the spots for visualization.

• 5.
  Examine the incubated cells by microscopy.

  • a.
    Microscopically inspect the incubated cells for abnormalities such as increased numbers of dead cells, contamination, etc.

Note: If desired, cell culture supernatants or cells can be carefully harvested and used for further analysis such as flow cytometry or ELISA. If this is not desired, proceed directly to the next step.

CRITICAL: Avoid scratching the bottom of the wells with the pipette tip as this will remove the coating and the bound antibodies, thus altering your results. Do not allow plates to dry completely between steps.

• 6.
  Discard the incubated cells and wash the plates.

  Note: “Washing” in the context of plates means immersing the plates in the appropriate wash buffer, discarding the buffer in one swift motion by inverting the plate, and then tapping the plates vigorously on a stack of paper towels as described in Step 2 of “before you begin.” This applies to all of the following steps, with slight variations.

  • a.
    Wash the plates once with Water - 0.1% Tween.
  • b.
    Wash the plates three times with 1x PBS – 0.05% Tween.
  • c.
    The third time, leave the 1x PBS – 0.05% Tween on the plate for 10 min.
  • d.
    Repeat Steps a–c) twice.

    CRITICAL: The total washing time should take at least 30 min.

    Note: Tween permeabilizes the cells; therefore, cells should be completely removed from the plates after this step.

    CRITICAL: Do not allow plates to completely dry between steps.

• 7.
  Incubate the plates with the AP-conjugated secondary antibody.

  • a.
    Dilute the appropriate secondary antibody to the required concentration or working dilution in 1x PBS – 1% gelatin – 1% Tween.
  • b.
    Discard the washing buffer from Step 6 of “step-by-step method details” in one swift motion by inverting the plate.
  • c.
    Using a multi-channel pipette, add 50 μL of the secondary antibody solution to each well.
  • d.
    Stack the plates above each other and cover the top plate with a lid.
  • e.
    Place the plates in plastic bags to prevent evaporation.
  • f.
    Incubate the plates for 1 h at 37°C.

Note: The specific secondary antibodies used here are AP-conjugated and have a working dilution of 1:4000 (see also key resources table). However, the protocol will also run with HRP-conjugated secondary antibodies when AEC (3-amino-9-ethylcarbazole) is used as the chromogenic substrate (see also problems 6 and 8).

CRITICAL: Avoid scratching the bottom of the wells with the pipette tip, as this will remove the coating and bound antibodies, thus altering your results.

• 8.
  Wash the plates.

  • a.
    Discard the secondary antibody solution in one swift motion by inverting the plate.
  • b.
    Wash the plates three times with 1x PBS – 0.05% Tween.
  • c.
    The third time, leave the 1x PBS – 0.05% Tween on the plate for 10 min.
  • d.
    Repeat Steps a–c) twice.

CRITICAL: The total washing time should take at least 30 min.

CRITICAL: Do not allow plates to completely dry between steps.

• 9.
  Incubate the plates with the ESA substrate working solution.

  • a.
    Discard the washing buffer from Step 8 of “step-by-step method details” in one swift motion by inverting the plate.
  • b.
    Using a multichannel pipette, add 50 μL of the freshly prepared ESA substrate working solution into each well.
  • c.
    Stack the plates above each other and cover the top plate with a lid.
  • d.
    Place the plates in plastic bags to prevent evaporation.
  • e.
    Incubate the plates for 12–24 h at 4°C, protected from light.

CRITICAL: Avoid scratching the bottom of the wells with the pipette tip, as this will remove the coating and bound antibodies, thus altering your results.

Note: It is also possible to incubate the plates for at least 4 h at 37°C or until spots are visible (protected from light). However, incubation at 4°C for 12–24 h usually results in more intense spots.

• 10.
  Wash the plates.

  • a.
    Wash the plates five times with distilled water.
• 11.
  Dry the plates.

  • a.
    Dry the plates at ∼20°C–22°C protected from light.

    Note: To speed up the process, the plates can be centrifuged briefly (upside down). Don’t use more than 35 g and ∼5 s.

  • b.
    Plates may be stored protected from light for as long as necessary.

Detection and quantification of spots

Timing: ∼0.5 h per plate

In this step, the plates are scanned and the spots in each well of the plates are counted, including a quality control of the counting parameters, using the ImmunoSpotR Series 6 Ultra-V Analyzer from C.T.L. and the C.T.L. software BioSpotR ImmunoSpot 5.1.36. This protocol describes the use of the software that has been found to work best in our hands for analyzing the enzymatic detection of Ig-secreting cells. The complete instructions can be found in the instrument manual. Further and more detailed instructions are often offered by the respective companies of the ELISpot devices in the form of specialized courses on how to use the instrument. As an alternative to the procedure described here, the software allows additional settings to be used. Of course, any other device that allows detecting enzymatically generated spots can be used. Next to the number of the spots, the spot size and intensity can also be measured to allow conclusions about the Ig-secretion of individual cells.

• 12.
  Scan the fully developed and dried ELISpot plates.

  • a.
    Turn on the ELISpot Reader.
  • b.
    Wait for the Switchboard to load and select the option “Full plate scan”.
  • c.
    Open the Dashboard and select your folder for data storage.
  • d.
    Select the correct capture format for your fabricate of 96-well flat bottom plates.

    Note: For the plates listed here, select “96 well BD Falcon”.

  • e.
    Select “Auto-Prealignment with Autocentering” for optimal camera placement during the scanning process.
  • f.
    Eject the drawer, place your plate in the space described, name your plate and load it.

    Note: You can choose to scan only certain rows, columns or individual wells of your plate. Therefore, use the “well selection” button and label the wells you want to scan.

  • g.
    Scan and eject your plate.
  • h.
    Repeat Steps e–g) until all plates have been scanned.
  • i.
    Return to the Switchboard.

    Note: The software will create a new folder for each scanned plate in the data storage folder you previously selected. You can resume the analysis at any time.

• 13.
  Set up the counting parameters and count the spots.

  • a.
    Open the Smart Count option in the counting menu on the Switchboard.
  • b.
    Load the plates to be analyzed with the same counting parameters such as all plates on which the same IgH-chain was analyzed.

    • i.
      Press the “Load plates” button.
    • ii.
      Check the gray box for each plate to be analyzed.
  • c.
    To set up your best fitting counting parameters, follow the steps described as indicated by the software (software Steps 2–4).

    Note: Wisely choose the parameters so as it fits the majority of the spots in each well. Normally, there is no perfect setting that fits every single spot. However, by setting the parameters in the best way possible and using them for each plate of the same experiment, the analysis remains as objective as possible.

    Note: Optionally you can 1. automatically remove fibers and 2. reduce the counted area per well (∼95%; can be normalized to 100% by the software) to avoid counting dirt or shadows from well walls.

  • d.
    Save the final parameters for future analysis in Step 3 of the software.
  • e.
    Start Auto-Count for all plates loaded in your analysis list in Step 5 of the software.
  • f.
    Return to the Switchboard after counting is completed.

    Note: The software will create a new folder for the counted plate in the data storage folder of the scanned plate. You can continue with the analysis at any time.

    Note: If you want to load the parameters from a previous analysis, select the “Counting Parameters” tab in software Step 2 and select your saved parameter settings. Proceed to fine-tune the parameters for each experiment as indicated by the software (software Steps 2–4).

• 14.
  Quality control of counted wells.

  • a.
    Open the Quality control option on the Switchboard.
  • b.
    Load the counted plates you wish to check by using the “Add plates” button and checking the gray box for each of the counted plate folders.
  • c.
    Start the quality control.

    Note: The program will display an overview of the first plate.

  • d.
    Look at each single well and check whether the spot numbers counted are correct.
  • e.
    If your well is completely blue or has too much blue background for reliable analysis, you can select “Set to TNTC” (too numerous to count) from the menu above your plate.

    • i.
      Double-click with the left cursor button on individual wells or entire columns/rows to set the wells to TNTC.

      Note: The results of these wells are not documented.

  • f.
    If your well is empty, but the software has counted dirt spots, you can set the well/row/column to zero by selecting the “Set to zero” option in the menu above your plate and then proceed as described in (e) above.

    Note: The results of these wells will be documented as zero.

  • g.
    If you are not satisfied with the results generated by the automated counting of the well, you can use the right cursor button to mark individual wells/rows/columns with an “R” for recount.
  • h.
    When all the wells that need to be recounted are marked with an “R”, check the box for “Recount/ QC individual wells” in the menu above your plate and open the first of these wells by double clicking the left cursor button.
  • i.
    Remove wrongly counted spots, such as dust spots.

    • i.
      Check the “Remove spots” box.
    • ii.
      Keep the check mark on the “Automatic removal” box.

      Note: You can also choose to remove the fibers at this point.

    • iii.
      Count the spots and follow the software’s instructions.
    • iv.
      Remove individual miscounted spots by double-clicking on the spot border with the left cursor button.

      Note: It is not possible to add spots manually.

  • j.
    When you are finished removing spots, press the right cursor button and confirm the results.

    Note: If you are not satisfied with the results at this point, start the process again.

  • k.
    Remove the “R” from the recounted well by clicking on it with the right cursor button.
  • l.
    In the upper right Batch Recount menu, check the boxes for “Apply for selected wells on the current plate only” and “Confirm count results for each well”.
  • m.
    Start the batch recount.

    Note: The software will now jump from each “R”-marked well to the next, allowing you to remove individual spots.

    Note: It may be necessary to interrupt the batch count by selecting “Do not confirm results” for a well, for example, because spots were mislabeled as fibers and removed from the analysis. The program will return to the individual well menu and ask you what you want to do. You will need to uncheck the “Fiber removal” box and start counting again. The software will return to the batch count and continue until you have confirmed the results of the last “R”-marked well.

  • n.
    If you are satisfied with the results, finish the quality control of the plate and move on to the next one in your list, or finish QC.

    Note: The software will create a new folder for the quality-controlled plate in the data storage folder of the scanned plate.

    Note: Whenever you manually change the results of a counted well during quality control, the software will document these changes (red numbers; see software manual for description) and display the changed results as green numbers in the lower right corner.

• 15.
  Quantify the spot numbers (Figure 1B).

  • a.
    Open the Excel sheet in the data folder of the quality-controlled plate to normalize your results.

    Note: Any numbers that were changed during quality control will be displayed in red on the Excel sheet.

  • b.
    Calculate the average for the technical triplicates of each dilution series.

    Note: You can do that by manual calculation or by Excel programming.

  • c.
    Select the dilution steps that give reliable results.

    Note: For this step, wells with well-defined spots and a dilution of approximately 1:3 when comparing the dilution series should be used. That process is similar to selecting the correct values for ELISA standards.

  • d.
    Back-calculate the counted numbers of each selected dilution step.

    • i.
      Multiply the numbers in the second row by 3, those in the third row by 3² etc.
    • ii.
      Calculate the average of these dilution steps.

      Note: The final result describes the number of spots/ASCs per 100000 seeded cells (when 100000 cells were plated per sample per well in the first row for analyzing total IgH-chain specific Ig-secreting cells) and can be used to compare samples and experiments.

      Note: If the assay was started with a different number of cells in the first well, we recommend using that number for normalization. Of course, in principle any fixed number can be used for normalization.

• 16.
  Quantify the spot size and intensity.

  Note: The Excel sheet in the QC plate data folder also provides an average spot size for each individual well. In addition, there is a detailed list of the number of spots with a specific size per well. These values can be used to estimate the amount of secreted antibodies per cell if the well was coated with an anti-Ig to detect the total number of ASCs (see also chapter “expected outcomes”).

  • a.
    Open the Smart count menu.
  • b.
    Load your counting parameters used for this specific plate.
  • c.
    Select the well you want to analyze and press the Spot Info button in the software Step 2.
  • d.
    Press the “Check spots” button.

    Note: The software will number the spots in the well.

  • e.
    Export the data to Excel.

    Note: In the Excel list, you will have several sheets with a lot of information, including a 3D image of the well where the spot size and intensity are visualized. You will also get a list and a diagram documenting the spot size and intensity for each individual spot in the well. These values can be used to estimate the antigen specificity of the secreted antibodies and the amount of secreted antibodies per cell, if the well was coated with an antigen (see also chapter “expected outcomes”).

    Troubleshooting 2, 3, 4, and 5.

Expected outcomes

The enzymatic assay described here will result in blue dots, with each of these spots describing an antibody-secreting cell (ASC) and the amount (and specificity) of antibodies secreted by it. As an example, mice with a C57BL/6 background were immunized and boosted (day 42) by intraperitoneal injection of 4-hydroxy-3-nitrophenyl-acetyl-keyhole-limpet hemocyanin (NP-KLH) in the adjuvant alum. The mice were sacrificed and their bone marrow cells were then isolated and analyzed for total Ig-secreting cells and antigen (NP)-specific Ig-secreting cells according to this protocol (Figures 2A and 2B). To demonstrate the specificity of the assay for detection of secreted antibodies, we also analyzed splenocytes from an untreated C57BL/6 wild-type mouse for total IgG- and IgM- secreting cells and compared the results with those from an untreated animal deficient in the Recombination activating gene 2 protein (RAG2-KO) and therefore lacking mature B- and T cells⁹ (Figure 2C). As expected, depending on the settings, we detected Ig-secreting cells and NP-specific Ig-secreting cells among the wild-type mice (Figures 2A–2C), but we did not detect any spots and thus antibody-secreting cells among the splenocytes of the RAG2-KO mouse (Figure 2C, RAG2-KO), demonstrating that the assay is indeed specific for ASCs. Further studies using the assay described here exactly as it is or adapted to the experimental setup have been published and can be used as examples.^{1,6,7,8,10,11,12}

Figure 2.

ELISpot assay to detect and quantify antibody-secreting cells in primary murine single cell suspensions

(A) To detect total antibody-secreting cell (ASC) numbers, plates are coated with an unlabeled anti-mouse Immunoglobulin (Ig) of the respective Ig-heavy chain isotype, unspecific bindings blocked by gelatin and incubated with 10 × 10⁴ bone marrow cells in the first row, followed by a serial 1:3 dilution for each row.

(B) To detect antigen-specific ASC numbers, mice were immunized with 100 μg 4-hydroxy-3-nitrophenyl-acetyl-keyhole-limpet hemocyanin (NP-KLH; 1 mg/mL) in alum intraperitoneally and boosted 42 days later with 50 μg NP-KLH in PBS intraperitoneally. Plates are coated with NP-BSA and incubated with 20 × 10⁵ bone marrow cells in the first row, followed by a serial 1:3 dilution from row to row.

(C) Detection of splenic total IgM- and IgG-secreting cells as described in A), comparing splenocytes from untreated wild-type mice and Recombination activating gene 2 protein (RAG2)-deficient mice lacking mature B- and T cells.

The assay allows to detect the number of the Ig-secreting cells of your interest (e.g., IgH-isotype, antigen-specificity) among the number of seeded cells. Therefore, the result will be the frequency of that specific ASC subpopulation in the analyzed organ. When analyzing total Ig-secreting cells, the plates are coated with an anti-Ig that binds to the Fc-part of the secreted antibodies. Therefore, the size of the spot indicates the amount of antibody secreted per cell and can be used to compare the secretion efficiency between cells and samples. When analyzing antigen-specific ASCs such as e.g., NP-specific ASCs, however, the wells are coated with the antigen itself (e.g., NP-BSA) and the secreted antibody binds to the antigen through its specific antigen-binding site in the Fab part. In this case, the distribution of the secreted antibodies on the well bottom under the ASCs also depends on the strength of the antibody-antigen binding and therefore on the antigen affinity of the secreted antibody. For this reason, the results are more complex but also provide more information about the secreted antibodies. To estimate the antigen affinity of these antibodies and the amount of secreted antibodies per cell, the ratio of the spot size to spot intensity must be determined.

The protocol can also be used with PVDF or nitrocellulose membrane plates.⁶ However, the use of classical ELISA plates, especially the highly adsorbent ones, shows comparable sensitivity in our hands, they are cheaper and easier to handle. In addition, we find it easier to distinguish spots from dirt, as the spots on ELISA plates tend to be larger. This also gives you a better idea of the binding affinity of the antibody, as the spot size varies more with changing antibody secretion per cell on ELISA plates than on membrane plates.

Limitations

The successful detection of (antigen-specific) antibody-secreting cells obviously depends on the immunological fitness of the mice. The genetic background, age and sex of the animal, as well as diet, housing conditions and the associated gut microbiota can influence the immune status of untreated mice or the immune response to a vaccine, respectively.^6,13 Furthermore, the combination of the organ of interest, the Ig-heavy chain isotype detected, and (if applicable) the vaccine and immunization strategy used is critical for a detectable number of (antigen-specific) antibody-secreting cells. For example, IgA-secreting cells are more likely to be detected in the bone marrow or the lamina propria of the small intestine than in the spleen.⁸ When determining antigen-specific antibody-secreting cells, it is also important to consider the appropriate analysis time point after the immunization of the animal. For example, it is more likely to detect NP-specific IgM-secreting cells in the spleen of mice 21 days after their immunization¹ than in the bone marrow 70 days after primary immunization.⁷ Comparable parameters lead to different results when analyzing human cell samples.

Technically, the detection of antigen-specific antibody-secreting cells depends on the availability of the antigen and its suitability for the coating of the plates. In this regard, the coating strategy is more likely to be successfully transferred from an established antigen-specific Ig ELISA to an ELISpot protocol. However, any kind of antigen can be tested for its potential usability. Furthermore, the detection of antigen-specific antibody-secreting cells requires a large number of cells per sample when all Ig-heavy chain isotopes are detected in technical triplicates. If challenged with the problem of too few cells, reducing the number of technical replicates may be a solution. However, this diminishes the reliability of the assay.

Troubleshooting

Problem 1

The viability of the single cell solution is too low (Step 8 of “before you begin”).

Potential solution

• •Homogenize tissues more carefully, e.g., use less pressure and speed while pressing them through the cell strainer.
• •Pipette more gently to reduce mechanical stress.
• •Reduce incubation times in the erythrocyte lysis buffer (or enzyme solution if enzymatic digestion is required).
• •Avoid prolonged storage of cell suspensions and seed them as soon as possible after isolation.

Problem 2

A huge amount of fibers/debris can be found in the wells during scanning (Step 12 of “step-by-step method details”).

Potential solution

• •Use lint-free tissue when drying the plates after each wash step.
• •Stick to the time frame of each wash step (30 min/Wash step in total) to properly remove cell debris.
• •Wash the fully developed plates in water again or gently blow the wells with a compressed air spray to remove remaining fibers.

Problem 3

No spots are detected (Step 12 of "step-by-step method details").

Potential solution

• •Your cells may have died due to stress during their isolation. Make sure to prepare your single cell suspensions carefully to avoid the induction of apoptosis or necrosis (See also problem 1). Check cell viability after incubation in the ELISpot plates.
• •You may have miscounted your cell concentrations and seeded fewer cells. Make sure that your cell counts are reliable.
• •Depending on the genetic background of your mouse strain and other factors such as the intestinal microbiota or the housing conditions, your mice may have different frequencies of antibody-secreting cells in various organs. You may have to increase the number of seeded cells per well to detect lower frequencies of antibody-secreting cells. It may be helpful to determine the number of plasmablasts and plasma cells in the organs of your mouse colony by flow cytometry analysis before performing ELISpot.¹⁰ However, this may need to be adjusted when isolating your cells by enzymatic digestion, as this treatment may clip surface proteins commonly used to identify antibody-secreting cell populations.⁸
• •When detecting antigen-specific antibody-secreting cells, e.g., after vaccination, make sure that your mice responded to the antigen at all, e.g., by checking antigen-specific immunoglobulin titers in the blood serum.
• •When detecting antigen-specific antibody-secreting cells, e.g., after vaccination, the antigen affinity of the secreted antibodies may vary between individuals, vaccination strategies, mouse strains, etc. Therefore, the format of the antigen used to coat the ELISpot plates must be carefully considered. For example, when immunizing mice with NP-KLH (in alum), the ELISpot plates are coated with NP-BSA as an antigen. However, companies typically offer several batches of NP-BSA that differ in the ratio of NP-molecules coupled to one BSA-molecule (load). The higher the number of NP-molecules coupled to one molecule of BSA, the more likely it is that antibodies, and therefore antibody-secreting cells, with a lower antigen-affinity will be detected.
• •When detecting antigen-specific antibody-secreting cells, e.g., after vaccination, make sure to wash your plates once with PBS before incubating the cells. The wash buffer in this step contains Tween 20 as a detergent. If Tween 20 is left in your plates when seeding the cells, it can cause cell death.
• •Make sure that all your buffers are prepared correctly, especially that they have the correct pH.
• •Make sure to combine the correct primary and secondary antibodies for each plate.
• •Make sure that your substrate solution is properly prepared, stored protected from light and is still colorless before use. If your substrate solution is already blueish before use, it has reacted prematurely and is no longer usable.

Problem 4

The whole well is blue or there is a lot of blue background that makes it difficult to identify spots (Step 12 of “step-by-step method details”).

Potential solution

• •You have seeded too many cells. As a result, the amount of antibodies secreted is so much, that it covers the entire well. See also problem 3 regarding the number of cells seeded.
• •You have stored your completely prepared cell suspensions (correct number of cells in complete medium, ready to seed) too long before seeding. As a result, the antibody-secreting cells have already secreted a large amount of antibodies into the medium. These antibodies then randomly bind to the coated well bottom and generate background.
• •Stick to the time frame of each wash step (30 min total per wash step) to properly remove cell debris.
• •Make sure that unspecific binding is blocked by performing Step 3 of "before you begin" correctly.
• •Make sure that the Substrate solution (BCiP concentration) was prepared correctly by checking the concentrations and amounts of reagents used in your notebook. If it is not clear that the solution has been prepared correctly, discard the solution properly and prepare it again.
• •Ensure that the unlabeled anti-Ig is used for coating and not the AP-coupled secondary antibody.

Problem 5

The spots are too faint, thus difficult to define (Step 12 of "step-by-step method details").

Potential solution

• •Depending on your mouse strain or their treatment, the amount of secreted antibodies per antibody-secreting cell may be lower than previous experience. Increasing the incubation time of your seeded cells may improve spot intensity.
• •When detecting antigen-specific antibody-secreting cells, after e.g., a vaccination, the antigen affinity of secreted antibodies may vary between individuals, vaccination strategies, mouse strains, etc. Therefore, the binding of the secreted antibody to the coated antigen may be too weak. For possible solutions, see also problem 3.
• •Make sure that you have used the correct concentration of primary and secondary antibodies and they are not expired or degraded.
• •Make sure your substrate solution is properly prepared, stored protected from light, and is still colorless before use. If the substrate solution is already blueish before use, it has reacted prematurely and is no longer usable.
• •Adjusting the protocol/parameters that define a spot using the ELISpot-reader software may still allow you to analyze your ELISpot plates, although the experiments should be repeated with an improved protocol adapted to your specific mouse colony and treatment procedure, and the data should be interpreted with caution.

Problem 6

You do not have enough cells per sample to perform your analysis in triplicates and for all Ig-heavy chain isotypes (Step 1 of "step-by-step method details").

Potential solution

• •You can reduce the number of technical replicates per sample; however, this reduces the reliability of the assay.
• •To increase the number of cells, samples from genetically identical, sex- and age-matched mice receiving the same treatment can be pooled. In this case, the use of sex-matched littermates would be ideal.
• •To increase the number of bone marrow cells, isolating lymphocytes from additional bones such as the humeri or the spine is an option.
• •It is possible to perform a Two-color-ELISpot analysis to detect, for example, two different Ig-heavy chain isotype secreting cells in the same well. Therefore, a horseradish peroxidase (HRP)-conjugated secondary antibody and AEC (3-Amino-9-ethylcarbazole) as a chromogenic substrate must be included in the protocol. However, using this protocol to perform a Two-color-ELISpot is more complicated, more often results in faint spots, and is more difficult to quantify. Note that commercially available kits show promising results using the Two-color enzymatic ELISpot approach but are very expensive.
• •Replacing the enzyme-linked antibodies with fluorochrome conjugated antibodies may increase the number of detectable parameters per well but requires a special ELISpot Reader equipped to detect fluorescence. However, the protocol described here has not been successful with fluorochrome conjugated antibodies. Note that commercially available kits show promising results using a multicolor fluorescent ELISpot approach but are very expensive and require special equipment for detection.

Problem 7

You want to analyze antibody-secreting cells in organs or cell cultures from other species, such as humans (Step 5 of "before you begin").

Potential solution

• •Theoretically, this protocol can be adapted for the cell samples of any species by simply exchanging the primary and secondary antibodies. However, this would require an establishment procedure. This protocol has already been successfully used for detecting human ASCs. See also the comments for problem 8.
• •When analyzing cell lines or in vitro activated B cells, the number of seeded cells must be adjusted.

Problem 8

You do not have access to certain reagents or an ELISpot Reader (all steps).

Potential solution

• •It may be possible to exchange individual reagents such as primary or secondary antibodies. If your laboratory has an established ELISA protocol and the secondary antibody is AP-conjugated, the antibodies used may also work for an ELISpot as described in this protocol. Using AEC (3-Amino-9-ethylcarbazole) as a chromogenic substrate for horseradish peroxidase (HRP)-conjugated secondary antibodies is also possible but usually results in faint spots.
• •Of course, similar chemicals or reagents from other companies may also work.
• •If you do not have access to an ELISpot Reader, it is possible to at least quantify the number of antibody-secreting cells using a microscope. However, we recommend that you label the plates in a blinded manner to eliminate subjective influences during counting. To further exclude subjective influences on the results, it may also be helpful to count the plates twice by two different people and use the mean of the results.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr. rer. nat. Katharina Pracht (katharina.pracht@fau.de).

Technical contact

Questions about the technical specifics of performing the protocol should be directed to and will be answered by the technical contact, Dr. rer. nat. Katharina Pracht (katharina.pracht@fau.de).

Materials availability

We did not generate new unique reagents.

Data and code availability

This protocol does not include a complete dataset generated and analyzed during a specific study. However, complete datasets are described in Bierling et al.,¹ Pracht et al.,⁷ or Daum et al.¹¹ This study did not generate original code.

Acknowledgments

We thank Heidi von Berg for animal caretaking. The graphical abstract and part of the figures were generated using SMART. The authors also thank the following funding sources: Interdisziplinäres Zentrum für Klinische Forschung (IZKF) Friedrich-Alexander-Universität Erlangen-Nürnberg and Deutsche Forschungsgemeinschaft (DFG, GRK2599).

Author contributions

C.B., F.R., and K.P. wrote the manuscript. S.R.M. and K.P. prepared the figures. J.C.-R. and K.P. conceived and developed the protocol. K.P. acquired funding and supervised the project.

Declaration of interests

The authors declare no competing interests.