Anti cd4 apc ef780

Mouse peripheral blood was obtained by retro-orbital venipuncture, collected in sodium citrate solution, washed, and stained as previously described [53 (link)]. Briefly, whole blood was incubated with red blood cell lysis buffer for 5–10 minutes and washed twice with PAB (1x phosphate-buffered saline, 1% bovine serum albumin, and 0.1% sodium azide) solution. Cells were probed with MHC-TSKB20 peptide tetramer complexes conjugated to Brilliant Violet 421 fluorophore and the following labeled antibodies: anti-CD44 PerCP-Cy5.5 (eBioscience, CA), anti-KLRG1 PE-Cyanine7 (eBioscience, CA), anti-CD8a FITC (Accurate Chemical), and anti-CD127 PE (eBioscience, CA). Cells were also stained with anti-CD4 APC-ef780 (eBioscience, CA), which was used as an exclusion channel. A minimum of 500,000 events were collected using a Cyan ADP flow cytometer (Beckman Coulter, FL) and analyzed with FlowJo Version X software (FlowJo, Ashland, OR). MHC-peptide complexes were provided as biotinylated monomers and/or fluorophore-conjugated tetramers by the NIH Tetramer Core Facility at Emory University (Atlanta, GA) and used for identification of CD8⁺ T cells specific to the TSKB20 (ANYKFTLV/Kb) epitope as described previously [26 (link)].

Peripheral blood mononuclear cells (PBMCs) from healthy controls were isolated by the Ficoll gradient (GE Healthcare, Zwijndrecht, The Netherlands). Cells were further isolated using the CD4+ T Cell Isolation Kit on an autoMACS Pro Separator, according to the manufacturer’s instructions (Miltenyi Biotec, Leiden, The Netherlands). To isolate the naïve subpopulation, total CD4⁺ T cells were stained with anti-CD4 APC-eF780 (eBioscience, Nieuwegein, The Netherlands), anti-CD27 BV510 (Biolegend, Amsterdam, The Netherlands), anti-CD25 PE, anti-CD127 Alexa Fluor-647 and anti-CD45RO PE/Cy7 (BD Biosciences, Vianen, The Netherlands) antibodies and purified on a BD FACSAria™ III cell sorter (BD Biosciences, Vianen, The Netherlands). Naïve CD4 ⁺ CD25⁻CD27 ⁺ CD45RO⁻ T cells were defined according to the gating strategy shown in Figure S5. Purity was consistently > 99%.