Anti cd90.2 53 2 1

Bone-marrow cells were incubated with biotinylated anti-CD3e (145-2C11; BD Biosciences) and anti-CD90.2 (53-2.1; BD Biosciences), followed by negative depletion with streptavidin beads (BD Biosciences). T-cell-depleted bone-marrow cells from Mazr^+/+Thpok^+/GFPLck-Cre or Mazr^f/fThpok^+/GFPLck-Cre mice (both CD45.2⁺) were mixed at a 1:1 ratio with CD45.1⁺ bone-marrow cells, and a total of 4 × 10⁶ cells were injected into the tail vein of lethally irradiated (8.25 Gy) CD45.1⁺ congenic mice. Eight weeks after transplantation, reconstituted mice were sacrificed, and iNKT cell development was analyzed.

Splenocyte stimulators from 2W-OVA.F1 mice were prepared and their red blood cells were lysed with ACK lysing buffer (Quality biological), followed by 30min incubation with anti-CD90.2 (53–2.1, BD Biosciences) to deplete T cells. Labeled T cells were depleted with two consecutive 35min incubations with rabbit complement (Cedarlane) at 37°C. >40× 10⁶ T-depleted splenocytes were then incubated overnight with 20μg/ml LPS. The following day, 1 × 10⁶ responder cells (Pan-T enriched splenocytes) were plated with 0.5 × 10⁶ stimulators (T-depleted APC’s) in triplicate in a 96-well plate (Corning) and incubated at 37°C overnight. Next, Golgi Plug (BD Biosciences) was added at 1:1000 and incubated for an additional 6h at 37°C. Live/Dead and extracellular staining were performed for 10min and 15min (respectively) on ice, and cells were then fixed with BD Cytofix/Cytoperm according to the manufacturer’s instruction (BD Biosciences). Finally, cells were stained for intracellular IFNg and TNFa and acquired via flow cytometry.