Tim 3 pe cy7

PBMC's were thawed in R20 medium, rested for 1 h at 37 °C, 5% CO₂, and stained with surface markers CD3 (Pacific Orange; Invitrogen, Thermo Fisher Diagnostics, Hemel Hempstead, Hertfordshire, UK), CD4⁺ (Qdot 605; Invitrogen), CD8⁺ (Pacific Blue, BD Biosciences), Live/Dead vivid marker (A750; Invitrogen), HLA-DR FITC (BD Biosciences), CD38 PECy7 (BD Biosciences), programmed death 1 (PD-1) APC (eBioscience, Thermo Fisher Diagnostics), Tim3 (PECy7; Biolegend, London, UK) and CD95 (APC; Biolegend), for 30 min at RT in the dark. Cells were then washed twice in PBS and fixed in 2% paraformaldehyde. Samples were acquired on an LSR II (Becton Dickinson, Wokingham, Berkshire, UK) flow cytometer within 12 h of staining and analysed using FlowJo version 8.8.6; FlowJo LLC, Ashland, Oregon, USA).

Analysis of the expression of cell surface and intracellular proteins in purified NK cells and in human carcinoma cell lines was performed by flow cytometry. Cells (1.0 × 10^{6 (link)}) were incubated with 1 μL per test of LIVE/DEAD Fixable Aqua (Thermo Fisher Scientific, Waltham, MA) in 1 × phosphate buffered saline (PBS) for 30 min at 4°C to accomplish live versus dead cell discrimination. Cells were then centrifuged, washed twice with cold PBS, and then stained with primary antihuman mAbs in 1 × PBS +1% BSA (Teknova, Hollister, CA) for 30 min at 4°C. Binding of NEO-201 to human carcinoma cell lines was detected by Pacific Blue-conjugated NEO-201 antibody (BioLegend, San Diego, CA). To detect the NK markers modulated by ALT-803, purified NK cells were labeled with following antibodies: CD56-PE (clone 5.1H11), CD16-PerCP-Cy5.5 (clone 3G8), Tim-3-PE-Cy7 (clone F38–2E2), NKG2D-BV421 (clone 1D11), CD107a-APC-Cy7 (clone H4A3), Granzyme B-FITC (clone GB11), PD-1-APC (clone EH12.2H7), and CD158d-APC (clone mAb 33) (BioLegend). After staining, cells were washed twice with cold PBS and examined using a FACSVerse flow cytometer (BD Biosciences, San Jose, CA). Analysis of cellular fluorescence was performed using BD FACSuite software (BD Biosciences). Positivity was determined by using fluorescence-minus-one controls.

Cell surface and intracytoplasmic marker expression were detected using a BD CantoII flow cytometer. For extracellular staining, anti-human CD56-FITC (eBiosciences, MA1-19129), NKG2D-PE (BD, 561,815), MICA/B-PE (Biolegend, 320,906), ULBP1-FITC (Invitrogen, MA5-38655), CD107a-APC (BD, 641,581), and TIM-3-PE-cy7 (Biolegend, 345,013) were used for extracellular staining. For intracellular staining, CAR-NK cells were fixed and permeabilized using a BD Cytofix/Cytoperm kit (BD Biosciences, Franklin Lakes, USA). following the manufacturer’s protocol, and anti-human IFN-γ-eFluor 450 (eBiosciences, 85-48-7319-42) was used for intracellular staining. Stained samples were acquired on a BD CantoII flow cytometry and analyzed with FlowJo software (Tree Star, Ashland, USA).