Cd3 efluor 450

Cells were stained with CD3 FITC (Cat# 11-0039), CD3 eFluor 450 (Cat# 48-0038), CD4 PE-Cyanine7 (Cat# 25-0049), CD4 PerCP-Cyanine5.5 (Cat# 560650, BD Pharmingen), Foxp3 APC (Cat# 17-4777), Foxp3 PE (Cat# 12-4776), CD45RA PE (Cat# 130-092-248, Miltenyi), CD45RA APC (Cat# 130-092-249, Miltenyi), CD62L APC (Cat# 17-0629), CD25 PE (Cat# 130-091-024, Miltenyi), CD25 APC-H7 (Cat# 560225, BD Pharmingen), CD8 PE-Cyanine7 (Cat# 25-0088), Perforin PE (Cat# 12-9994), granzyme B PE (Cat# 12-8899), CCR8 APC (Cat# FAB1429A, R&D System), CD4 FITC (Cat# 11-0049), CD45 APC (Cat# 17-9459), CD14 PE (Cat# 12-0149), CD45RO PE (Cat# 12-0457), CD45RO eFluor 450 (Cat# 48-0457), CD127 APC (Cat# 17-1278). PITPNM3 antibody (Cat# NBP1-31070, Novus) was labeled with APC by Abcam APC Conjugation Kit (ab201807, Abcam) according to the manufacturer's instructions. For the intracellular stain, cells were pretreated with Intracellular Fixation and Permeabilization kit (Cat# 88-8824) according to the manufacturer's instructions. All the reagents were from eBioscience unless indicated otherwise. Cells were subsequently analyzed by multicolor flow cytometry (Gallios, Beckman Coulter, China).

K562 (HLA-A2) or Granta-519 cells were loaded with peptide overnight at 37 °C in a 5% CO₂ incubator. Effector cells were stimulated with target cells at an effector-to-target (E:T) ratio of 1:2 for 5 hours at the same conditions as above. Conjugated CD107a was added to the cells prior to incubation. Irrelevant or no peptide served as a negative control.
The following antibodies were used: Vβ3- FITC (Beckman Coulter-Immunotech SAS, France), CD3-eFluor450, CD56- eFluor, CD107a-PE-Cy5, TNFα-PE (BD Biosciences, USA), IL2-APC, IFNγ-FITC (eBiosciences, ThermoFischer). Cells were washed in flow buffer (FB, phosphate buffered saline (PBS) with 2% human bovine serum albumin (BSA) and 0.5 µM EDTA). For dextramer and antibody staining, cells were incubated for 30 minutes at room temperature (RT) with the recommended dilution in FB. If fixed, cells were incubated in FB containing 1% paraformaldehyde. For intracellular staining Perm/Wash Buffer was used (BD Biosciences) according to manufacturer’s protocol. All antibodies were purchased from eBioscience, USA, except where noted. Cells were acquired on a BD FACSCanto II flow cytometer and the data analyzed using FlowJo software (Treestar Inc., Ashland, OR, USA). Plotting and statistical analysis were performed using GraphPad prism software (La Jolla, CA, USA).