Step-by-step Memory B cell stimulation

Get your memory B cells ready for ELISpot/FluoroSpot with our StimPacks (included in all B cell ELISpot/FluoroSpot kits).

In order to induce secretion of detectable amounts of antibody, memory B cells require polyclonal stimulation. We searched far and wide and tested all sort of combinations of different stimuli and found a combination of the TLR7/8 agonist R848 and recombinant IL-2 worked great for this purpose. Thus the StimPack: Memory B cells was born! 

 

B cell stimulation with R848 and IL-2

R848 and IL-2 are both immunostimulatory agents that can stimulate activation and proliferation of B cells. R848 is a synthetic immune adjuvant that activates immune cells, including B cells, through the toll-like receptor 7 and 8 (TLR7/8). When R848 binds to the TLR, it activates the immune cell and stimulates the production of proinflammatory cytokines, enhancing the immune response. IL-2 is a cytokine that is produced by activated T cells and natural killer cells and plays a key role in the immune response by promoting the proliferation and activation of immune cells, including B cells. Together, R848 and IL-2 can stimulate memory B cells into antibody secreting cells for use in ELISpot and FluoroSpot applications. Read up on how you can use ELISpot and FluoroSpot to characterize B cell responses here!

Step-by-step protocol - memory B cell stimulation

Learn how to stimulate the memory B cells in your PBMC sample prior to use in ELISpot and FluoroSpot. Stimulation can take anywhere from 3-5 days and optimal time points should be determined for your specific application.

1. Reconstitute the included recombinant IL-2
   • Add 1 ml of PBS to obtain 1 µg/ml human IL-2 (or 0.5 µg/ml mouse IL-2), and leave for at least 15 minutes at RT.
   • Vortex and use immediately or store in aliquots at -20°C.
2. Thaw and wash cryopreserved cells in PBS and resuspend in fresh complete media. Alternatively, freshly prepared cells can be used.
3. Count and dilute cells to 2 million cells/ml and plate 1 ml per well to a sterile 24 well flat bottom cell culture plate. Alternatively, add 1 ml to sterile tubes (e.g., 5 ml polypropylene round bottom Falcon tubes, cat. No 352063).
   
4. Prepare the R848 and rIL-2 stimulation cocktail to be added to each well or tube, resulting in a final concentration of R848 (1 µg/ml) and rIL-2 (10 ng/ml) for stimulation.
   • Example: for 8 wells of cells prepare 8 ml of stimulation cocktail (plus extra).
   • 10 ml stimulation cocktail needed = 20 µl R848 (1 mg/ml) + 200 µl IL-2 (1 µg/ml) + 10 ml complete media.
5. Add 1 ml of stimulation cocktail to each well or tube. For plates, fill empty surrounding wells with sterile H₂O.
   
6. Wrap in aluminum foil and incubate 3-5 days at 37 °C 5% CO₂ and humidity.
7. Following incubation, resuspend the cells and wash 2x in warm complete media.
8. Resuspend to appropriate concentrations for use in ELISpot/FluoroSpot.
    

Tips:

• Calculate how many cells you’ll need for your ELISpot/FluoroSpot and stimulate at least twice as many to be sure you’ll have enough for your assay.
• Check each well that there is an even dispersal of cells in solution prior to incubation if culture plate is used.
• After 3-5 days of stimulation, the cells should form small islands along the bottom of the tissue culture plates. This is a positive sign of successful stimulation!
• The media should not turn bright yellow after incubation for 3-5 days. If it does, this indicates a lower pH in the culture media, due to too many cells initially added. This will not be beneficial for cell viability and Ig-production.