Anti mouse cd45rb

Purified CD4+T cells (CD4⁺CD25⁻CD45RB^hi) from spleens and lymph nodes of Mina KO or WT litter mate control mice were FACS sorted on a Reflection (i-Cyt, Champaign, IL) following staining with anti-mouse CD4, anti-mouse CD45RB and anti-mouse CD25 (eBioscience) were differentiated in to Th1 or Th2 as per previously published conditions [54 (link)]. Briefly, CD4+T cells were stimulated in anti-CD3 coated plates (5ug/ml), anti-CD28 (1ug/ml) in presence of rIL12 (20ng/ml) and anti-IL4(10ng/ml) for Th1 and anti-IFNγ (10ng/ml), rIL4 (100ng/ml) and anti-IL12 (10ng/ml) for Th2 differentiation. Cells were cultured for three days and restimulated with PMA (50 ng/ml) and ionomycin (1 μg/ml) in presence of monensin to test for intracellular cytokine IFNγ and IL4.

CD4⁺T cells (CD4⁺CD25⁻CD45RB^hi) and B cells (CD19⁺) from spleens and lymph nodes of Mina KO or WT litter mate control mice were FACS sorted on a Reflection (i-Cyt, Champaign, IL) following staining with anti-mouse CD4, anti-mouse CD45RB and anti-mouse CD25 (eBioscience) for T cells and anti-mouse CD19 for B cells. CD4⁺T cells (1x10⁵ cells/ml) were stimulated for 72h with soluble anti-CD28 (1ug/ml, eBioscience) in 96 well flat bottom plates coated with graded amounts of anti-CD3 (eBioscience). B cells (1x10⁶ cells/ml) were stimulated in 96 well flat bottom plates with graded amounts of LPS (O111:B4, Sigma) for 72 h. Cultures were pulsed with 1 μCi of [³H]-thymidine for the final 8h of assay and harvested with a Packard harvester. The counts per minute (cpm) were determined using a Packard Matrix 96 direct counter (Packard Biosciences).