Easysep cd8 negative selection kits

CD8⁺ polyclonal or OT-I T cells were isolated from lymph nodes of 6–8-week-old mice using EasySep CD8 negative selection kits (STEMCELL Technologies). B cells were isolated from spleens of 6–8-week-old mice using EasySep B cell negative selection kits (STEMCELL Technologies). In specified experiments, B cells were treated with 1 μg/ml LPS (Sigma-Aldrich) for 5 h before use. BMDCs were generated from treating cultured bone marrow cells with GM-CSF (granulocyte-macrophage colony-stimulating factor) for 7–11 days. IL-4 was added for the last 2 days with 1 μg/ml LPS stimulation 6–24 h before use. BMDCs and B cells were incubated with 1–100 ng/ml SL8 OVA peptide (SIINFEKL) (Anaspec), or variant peptides N4, T4, Q4H7, V4 (gift from E. Palmer and D. Zehn) for at least 30 min at 37°C and washed 3 times.

Inguinal, axillary, brachial and mesenteric lymph nodes (LN) or spleens were isolated from CD45.1 OT-I or P14 LCMV mice. LN and spleens were meshed through 70μm filters and treated with ACK red blood cell lysis buffer. CD8+ T cells were purified using EasySep CD8 negative selection kits (Stemcell Technologies). 1×105 (for >14 day read-out) – 2×106 T cells (for day 4 read-out) were adoptively transferred through retro-orbital injection in 100μl PBS.
For the comparison of OT-I T cells and p14 LCMV T cells, mice received a 1:1 mix of both T cells in 100μl of PBS through retro-orbital injection. The following day mice were inoculated with a bolus of CFA containing gp33-peptide (50μg/mouse; Anaspec) and SL8/SIINFEKL peptide (50μg/mouse; Anaspec) subcutaneously, to sustain both T cell populations.