GSM6890118: TFH_Tet2-/- #4; Mus musculus; RNA-Seq
At sacrifice, single cell suspensions were prepared from the hematopoietic organs (spleen and LNs). The tissues were crushed with a 1 mL syringe plunger through a 40 µm nylon mesh in cold buffer (phosphate-buffered saline (PBS 1X) without MgCl2 and CaCl2, supplemented with 0.5% bovine serum albumin, 2mM EDTA pH8.0). After centrifugation, cells are resuspended in red blood cell lysis buffer for 8 minutes at room temperature. After wash, cells were counted on a ViCell Counter and resuspended at the appropriate dilution for further experiment. CD3 T cell were isolated using spleen and LN isolated by negative selection using biotin coupled antibodies: anti-CD19 (Biolegend #115504) anti-B220 (Biolegend #103204), anti-Ter119 (Biolegend #116204), anti-NK1.1 (BioLegend #108704), anti-CD49b (Biolegend #108904), anti- Ly-6G/Ly-6C (Biolegend #108404), anti-CD11b (Biolegend #101204), anti-CD11c (BioLegend #117304), followed by Streptavidin Particles Plus - DM (BD, #557812) incubation according to manufacturer's instructions. Then, Tfh cells (viable CD4+ CXCR5+ PD-1+) were sorted using a FACSAria II or FACSAria Fusion fluorescence-activated cell sorter (BD Biosciences, purity check>98%). 250.000 Tfh were collected and extracted using the AllPrep DNA/RNA Micro Kit (Qiagen #80284), following the manufacturer's instructions.