Cell sample preparation T cell ELISPOT and FluoroSpot assay
Notes:
- Both fresh and cryopreserved cells can be used for ELISPOT and FluoroSpot analysis. Important is that the cells are viable. For more information see: Guidelines for cell collection and handling.
- For detailed information on recommended stimuli and incubation times see: Guidelines for stimuli, cell concentrations and cell incubation times.
- It is recommended to test the samples in triplicate and in serial dilutions in the ELISPOT and FluoroSpot procedure. (Since a certain cell number is needed for sufficient stimulation, the assay does not always show linearity in serial dilutions.)
- Although a high cell density increases the likelihood of contact between stimulating and responding cells during stimulation, no more than 3x105 cells/well should be used in the ELISPOT or FluoroSpot plate. Higher concentration of cells will cause multiple cell layers, resulting in poor spot formation.
Stimulation with small peptides (8-12 amino acids) or determine a memory response
Small peptides can directly be presented by antigen presenting cells (APCs) to T cells. Consequently, such peptides and memory responses can be used in the ELISPOT and FluoroSpot assay without a preincubation step. After cells are counted, suspended cells in cell culture medium with an appropriate stimulus. For antigen-specific stimulation, 1-3x105 cells/well is recommended. For polyclonal stimulation, the recommended cell concentration per well should be reduced to 2x102 – 1x105 cells/well. The volume of the cell suspensions in the 96-well ELISPOT or FluoroSpot plate is 100 µl/well.
Preincubation of cells
A 24-48 h preincubation step at high cell density (>106) may be required when full-length proteins or long peptides are used for in vitro re-stimulation. These long antigens must first be internalized, processed and presented by APCs via MHC class I/II molecules before they can stimulate cytokine (or other effector molecule) release by T cells. The high number of cells enhances the probability of contact between stimulating and responding cells. Omitting this step leads in most cases to a significant lower frequency of spot forming cells.
A preincubation step can also be useful when the analyte of interest requires longer incubation.
For preincubation, gently suspend cells in cell culture medium with an appropriate stimulus at 4x106 cells/ml in a tissue culture plate and incubate 24-48 h (37 °C with 5% CO2 and 100% humidity). 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.
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 cytokines or other effector molecules produced during the preincubation step. Thereafter, cells are counted and suspended in cell culture medium with the same stimulus as used during preincubation at 1-3x105 cells/well (antigen-specific responses). For polyclonal stimulation, the recommended cell concentration per well should be reduced to 2x102 – 1x105 cells/well. The volume of the cell suspensions in the 96-well ELISPOT or FluoroSpot 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.
Culture medium AIM V (Thermo Fisher Scientific cat. no. 31035-025) supplemented with 100 units/ml penicillin and 100 μg/ml streptomycin is an alternative cell culture medium for the ELISPOT procedure without a preincubation step.