After sorting the CD3 positive cells from the spleen, I stimulated them in a p96 previously coated with anti-CD3 and anti-CD28 for 3 hours, and then I cleaned the wells with PBS. Does anybody know how long the coating is useful for in these conditions? Is it better to keep the plate with the coating until use, or keep them in PBS?
Thank you Qiang for your answer. Just one last thing: if the plate has been in the incubator overnight and then kept in the cold room (with PBS after the coating), for how long do you think I can still use it?
Thank you in advance.
Hi! I stimulated murine CD4+ and CD8+ T cell in one of my experiment. The details are below: CD4+ and CD8+ T cells were stimulated by incubating the cells in wells of a microtiter plate that had been precoated with an anti-CD3 antibody (4 µg/mL and 8 µg/mL respectively, overnight in PBS at 4 degree, then wash the well with fresh PBS 2 or 3 times) and in the presence of a soluble anti-CD28 antibody (2 µg/mL, it was added to the cell the next morning). For your last question, I have tested before. But I think 3-5days is no problem. Good luck with you!
hi,
the recommended protocol for CD3 (clone 145-2c11) bound coating is 2h at 37c or at +4c overnight. I have left the plate at 37c overnight. Now, i have washed the plate 3 times with PBS and stored at +4c for additional 24 hours. do you think it will be ok to use the plate? does anyone have such experience?
thank you,
- Badges
- Science topic