Cd62l fitc

The following mAbs were used for staining and flow cytometric measurements: CD3-VioGreen (BW264/56), CD4-allophycocyanin-Vio770 (VIT4), CD8-PE-Vio770 (BW135/80), CD14-peridinin chlorophyll protein (PerCP) (TÜK4), CD20-PerCP (LT20), CD27-FITC (M-T271), CD28-FITC (15E8), CD45RA-VioBlue (T6D11), CD45RO-FITC (UCHL1), CD62L-FITC (145/15), CD107a-PE (1D4B), CD127-FITC (MB15-18C9), CD137-allophycocyanin, CD137-PE (4B4-1), CD154-VioBlue (5C8), CD178-PE (NOK-1), CD197 (CCR7)-allophycocyanin, CD197 (CCR7)-PE (150503), CD279-allophycocyanin (PD1.3.1.3), anti-IL-2-PE-Vio770 (N7.48A), anti-IL-4-PE (7A3-3), anti-IL-17-FITC (CZ8-23G1), anti-IFN-γ-FITC, anti-IFN-γ-PE-Vio770 (45-15), anti-TNF-α-PE, and anti-TNF-α-VioBlue (cA2) (all Miltenyi Biotec).
Soluble biotinylated pHLA-A*0201 molecules loaded with WT1₃₇ (VLDFAPPGA), WT1₁₂₆ (RMFPNAPYL), WT1₁₈₇ (SLGEQQYSV), WT1₂₃₅ (CMTWNQMNL), or cytomegalovirus (CMV) pp65₄₉₅ (NLVPMVATV) were produced as described previously.14 (link) Tetramerization was achieved by binding to streptavidin-PE or streptavidin-allophycocyanin (both BioLegend, San Diego, CA). In each case, 2·5 × 10⁶ cells were stained with 1 µg/ml tetramer for 45 min at 4°C.
Data were acquired using a MACSQuant flow cytometer and analysed with MACSQuantify software (both Miltenyi Biotec).

Transduced cells were stained with fluorochrome-conjugated monoclonal antibodies for 15 min at 4°C. The cells were washed twice with 1x PBS supplemented with 1% FBS. For cell surface marker analysis, the following antibodies were used: CD56-PE (HCD56/Cat#130-114-551), CD4-FITC (OKT4/130-114-531), CD8-APC (SK1/130-110-679), CD45RO-APC (UCHL1/130-113-556), CD62L-FITC (DREG-56/130-112-077), CD25-APC (BC96/130-113-284) (Miltenyi Biotec, Bergisch Gladbach, Germany), PD1-FITC (EH12.2H7/329904), LAG-3-FITC (11C3C65/369308), TIM-3-APC (F38-2E2/345012), and CD3-PerCP (clone OKT3/317336) (BioLegend, San Diego, CA, USA). The memory phenotypes were defined as naïve (TN; CD3⁺CD45RO⁻CD62L⁺), effector memory (TEM; CD3⁺CD45RO⁺CD62L⁻), central memory (TCM; CD3⁺CD45RO⁺CD62L⁺), and terminal effector (TE; CD3⁺CD45RO⁻CD62L⁻) T cells. For transduction efficiency of transduced cells, AF647-conjugated anti-human IgG (H+L) (Jackson ImmunoResearch/109-607-003) was used to detect CAR expression. Flow cytometry was performed using a BD Accuri™ C6 Plus Flow Cytometer (BD Bioscience), and data were analyzed by the FlowJo V10.7.1 software (FlowJo).