my b cell culture looks marvelous. flow sort for resting b cells, then plate on NIH3T3tCD40L cells (200K cells per well of a 6 well plate). split every 3rd day. culture in DMEM F12 supplemented with insulin and IL-4. you end up with activated b cells expressing CD62L, CD83, CD86. they grow like gangbusters!
now to use this as antigen-presenting cells to detect antigen-specific T cells........
and it is spring here in Seattle, beautiful night last night. had a moonlight picnic down at The Cut.
check out this expansion of antigen-specific t cells. bulk t cells from whole blood were stimulated with cultured B cells (described above) pulsed with 9mer and 40mer peptides (from HIV envelope gp120....the V2 "crown"). unpulsed cultured B cells as a control. the t cells were cultured with the B cells for 8 days (supplemented with IL-2,7,15 days 3,5, and 7). at the end the t cells were stained with a tetramer containing the red peptide sequence. i used HLA matched HIV seronegative t cells as a control to test for de novo
reactivity (there was none...pretty clean "background"). we are now writing a grant to get more wayne cashman so we can do more experiments. we think we will be able to detect t cells responses in vaccine trials with this assay and "map" t cell responses in natural history cohorts.
Keep up the good work Skidoo! Good luck with your grant.