Tim 3 fitc

PBMCs from subjects were resuspended in PBS buffer and were incubated with directly conjugated antibodies for 30 min at 4°C. The cells were washed with 1× PBS before flow cytometry analysis. Antibodies used included anti-human CD3-BV786, CD19-BVAPC-H7, CD16-BV711, CD69 PE-CF594, CD57-BV421, PD-1-APC, CD70-PE, CD160-AF488, 2B4-AF700, CTLA-4-PE, TIM-3-FITC, 7-AAD (BD Biosciences, San Diego, CA, USA), CD56-BV510, BTLA-PE-CY7, CD39-APC (BioLegend, San Diego, CA, USA), TIGIT-PE-Cy7 (Ebioscience, San Diego, CA, USA), along with the corresponding isotype controls. NK cells were gated as CD3⁻CD14⁻CD19⁻CD56⁺CD16⁺, CD3⁻CD14⁻CD19⁻CD56⁺CD16⁻, or CD3⁻CD14⁻CD19⁻CD56⁻CD16⁺. Data were acquired with the LSR Fortessa flow cytometer (BD Biosciences) and analyzed with FlowJo software version 10.5 (Tree Star, Ashland, OR, USA).

Briefly, freshly collected whole-blood samples were incubated for 30 min at room
temperature with fluorochrome-labeled monoclonal antibodies: TIM-1-PE, TIM-3-FITC and
TIM-4-PE (BD Biosciences, USA). The red blood cells were lysed for 15 min and, after
washing twice with PBS, the remaining cells were assayed within 30 min by
fluorescence-activated cell sorting (FACSCalibur, BD Biosciences). Flow cytometry
data were collected and analyzed using the CellQuest software (BD Biosciences).