Antibody-secreting B cells (plasma cells) were assessed by B cell ELISPOT assay according to the manufacture’s protocol (MabTech, Nacka Strand, Sweden). Briefly, ELISPOT plates were coated with capture anti-IgG or anti-IgM antibody (BD Pharmingen), and then spleen and bone marrow cell suspensions in 5% FBS/complete RPMI were added to each well and incubated for 24 h at 37°C in 5% CO2. Plates were extensively washed to remove the cells and biotinylated anti-IgG or anti-IgM antibody was added followed by Streptavidin-ALP and substrate (BCIP/NBT; MabTech). After color development, total spots per well were quantified using an ImmunoSpot Series 2 Analyzer (Cellular Technology Ltd., Shaker Heights, OH). Priming of donor-reactive T cells to IFN-γ–producing cells was quantified by ELISPOT assay as previously described (19 (link), 20 (link)).
Protocol detail
Quantifying Antibody-Secreting B Cells
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Anti antibody
Anti igg
Antibody secreting cells
Assay
B cell
Bone marrow cell
Cells
Donor
Elispot
Ifn γ
Plasma cells
Spleen
Spots
Streptavidin
T cells
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Variable analysis
independent variables
- Presence or absence of antibody-secreting B cells (plasma cells)
- Number of IgG-secreting cells
- Number of IgM-secreting cells
- Number of IFN-γ-producing donor-reactive T cells
- Incubation time of 24 hours
- Incubation temperature of 37°C
- Incubation atmosphere of 5% CO2
- Cell culture medium (5% FBS/complete RPMI)
- Positive control: ELISPOT plates coated with capture anti-IgG or anti-IgM antibody
- Negative control: Not explicitly mentioned
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