Anti cd3 fitc ucht1

PBMCs were isolated with the Ficoll-Paque density gradient centrifugation protocol. CD4⁺ and CD8⁺ T cells were isolated from PBMCs with Dynabeads CD4⁺- and CD8⁺-positive selection kits (Invitrogen), respectively. For isolation of EM, EMRA, CM, and SCM memory subpopulations, we stained PBMCs with the following antibody mix: anti-CD3-FITC (UCHT1, eBioscience), anti-CD45RA-eFluor450 (HI100, eBioscience), anti-CCR7-APC (3D12, eBioscience), and anti-CD95-PE (DX2, eBioscience). Cell sorting was performed on FACS Aria III, and all four isolated subpopulations were lysed with Trizol reagent immediately after sorting.

We isolated PBMCs from the blood using standard Ficoll-Paque protocol. CD4 and CD8 fractions were isolated with CD4/CD8 Positive Selection Dynabeads Kits according to the manufacturer’s protocol. For isolation of memory subsets, we stained PBMCs with the mix of antibodies: anti-CD3-FITC (UCHT1, eBioscience), anti-CD45RA-eFluor450 (HI100, eBioscience), anti-CCR7-AlexaFluor647 (3D12, BD Pharmingen), anti-CD95-PE (DX2, eBioscience). Four subsets of cells were sorted into RLT buffer (Qiagen) on BD FACS Aria III: EM (CD3+CD45RA-CCR7-), EMRA (CD3+CD45RA+CCR7-), CM (CD3+CD45RA-CCR7+), Tscm (CD3+CD45RA+CCR7+CD95+). HLA-A02 dextramer loaded with the NS4B_241-222 peptide (LLWNGPMAV) from YFV17D (Immudex) was used for epitope-specific T-cells isolation. Cells were stained with NS4B-dextramer-PE, anti-CD3-eFluor450 (UCHT1, eBioscience), and anti-CD8-FITC (SK1, eBioscience) according to the manufacturer’s protocol. RNA was isolated using standard TriZol protocol (for bulk PBMCs, CD4 and CD8, NS4B-specific and negative fractions) or RNAeasy Micro Kit (Qiagen) (for memory subsets). The amount of RNA was measured on Qubit 2.0 (Invitrogen). Information about all antibodies and commercial kits could be found in Key Resources Table.