Cell sample preparation B cell ELISPOT assay

Cell sample preparation B cell ELISPOT assay

Notes:

• Both fresh and cryopreserved cells can be used for ELISPOT analysis. For more information see: Guidelines for cell collection and handling. 
• Optimal concentrations and incubation times should be experimentally determined, especially for working with heterogenous cell populations.
• It is recommended to test the samples in triplicate and in serial dilutions in the ELISPOT procedure.
• No more than 3x10⁵ cells/well should be used in the ELISPOT plate. Higher concentration of cells will cause multiple cell layers, resulting in poor spot formation. 
• Stimulation with IL-4 and anti-CD40 (stimuli of the IgE B cell ELISPOT) induces immunoglobulin class switching (Hasbold J. et al. 1998). Results obtained from cells stimulated with these reagents may therefore not be representative for the situation in vivo.

Preincubation of cells for activation of memory B cells

Quiescent memory B cells do not produce antibodies in significant quantities in vivo. Therefore, a preincubation step of several days in vitro at high cell density (>10⁶) and with appropriate polyclonal stimuli is required to activate these memory B cells. Since memory B cells also expand during activation, the ratio of both antigen-specific antibody secreting cells (ASCs; Assay II) and total number of ASCs (Assay I) is used to determine memory B cell responses (see Flow diagram B cell ELISPOT assay).

For preincubation (37 °C with 5% CO₂ and 100% humidity), suspend cells in cell culture medium with the appropriate stimuli in a tissue culture plate. Use a minimum of 1 ml/well in a 24-well plate, 0.5 ml/well in a 48-well plate or 100 μl/well in a 96-well plate.

Recommended preincubation times, cell concentrations and stimuli

The above mentioned incubation times are guidelines. If other cell types are used, other incubation times may have to be considered.

After preincubation, the non-adherent cells are collected and washed twice (8 min, 200 x g, 20-26 °C) with fresh cell culture medium without stimuli or fetal calf serum. This will avoid the carryover of antibodies produced during the preincubation step. Thereafter cells are counted and suspended in cell culture medium without stimuli at 1-3x10⁵ cells/well (antigen-specific responses [Assay II] and background response). For determination of the total number of ASCs (Assay I), the recommended cell concentration per well should be reduced to 2x10³ – 1x10⁵ cells/well. The volume of the cell suspensions in the 96-well ELISPOT plate is 100 µl/well.

Recently in vivo activated B cells

In vivo activated B cells (e.g. after vaccination) may produce immunoglobulins. The peak of the immunoglobulin production is 3 to 9 days post-vaccination. This respons can be directly analyzed in the ELISPOT assay (Assay II) without a preincubation step.

After cells are counted, suspended cells in cell culture medium without stimuli at 1-3x10⁵ cells/well (antigen-specific responses [Assay II] and background response). The volume of the cell suspensions in the 96-well ELISPOT plate is 100 µl/well.

Recommended cell culture medium

RPMI-1640 supplemented with 2 mM L-Glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin and 10% fetal calf serum (FCS):

• RPMI-1640: Thermo Fisher Scientific cat. no. 52400. 
• L-Glutamine (200 mM): Thermo Fisher Scientific cat. no. 25030-081. 
• Penicillin-Streptomycin (100x): Thermo Fisher Scientific cat. no. 15140-122. 
• FCS (should be selected on low background staining): Thermo Fisher Scientific cat. no. 26140. 

To B cell ELISPOT assay guidelines