Cd44 pecy5

One million cells of each tumor were transferred to a 96-well round-bottom, micro test plate and pelletized at 1500 rpm for 5 min (Beckman-Coulture Allegra X-14 Centrifuge). A fixable viability dye (eBioscience, eFluor 780) was used to identify live cells. The following antibodies were used for surface staining: CD3 APC (Biolegend, Clone: 17A2), CD4 BV510 (BD Bioscience Clone RM4–5), CD8a eFluor 450 (eBioscience, Clone: 53–6.7), CD279 (PD-1) FITC (eBioscience, Clone: J43), CD44 PECy5 (eBioscience, Clone: IM7), CD335 PECy7 (Biolegend, Clone: 29A1.4), CD11b AF488 (Biolegend M1/70), F4/80 BV421 (Biolegend BM8), CD206 PE (Biolegend C068C2), CD86 APC (Biolegend GL-1). Briefly, cells were stained with Fc blocking antibodies (TruStain FxX Biologend) for 10 min at 4 °C followed by cell surface antibodies in FACS Buffer (PBS with 2% FBS) for 30 min at 4 °C. Cells were pelletized at 1500 rpm for 5 min before re-suspending in 200 μL of FACS Buffer. Expression of T cell surface markers was measured by fluorescence cytometry (MACSQuant, Miltenyi Biotec) and analyzed by FlowJo software (Tree Star Inc.).

We used the following antibodies: CD44-PE-Cy5 (1:100) (eBioscience), CD15-APC (1:5) (BD), CD133/2-PE (1:50) (Miltenyi Biotec). Annexin V-FITC Apoptosis Detection Kit was obtained from eBioscience. Staining was quantified by flow cytometry (Cytek DxP10).