Cd3 buv496

The following fluorochrome-conjugated anti-human mAb were used for flow cytometry studies: LAG3 BV650, TIGIT PE-Cy7, CTLA4 PE, CD25 PE-Dazzle 594, TIM3 PerCP-Cy5.5, CD19 Alexa Fluor 700, CCR4 BV421, ICOS PE-Cy7, CCR5 PE-Dazzle 594, HLA-DR PE, CCR7 FITC, CD38 BV711, PD-L1 BV711, CCR6 Alexa Fluor 700, PD-1 BV421, CD40L BV605, perforin PE-Dazzle 594, and CD8 PerCP from BioLegend (San Diego, CA); CD3 BUV496, CD4 APC-Cy7, CD4 APC-H7, CD127 BV605, Ki-67 PerCP-Cy5.5, PD-1 BV650, CXCR3 BV605, CD69 BV650, IL-2 BV711, CXCR5 Alexa Fluor 647, IFN-γ PE-Cy7, TNF-α FITC, granzyme B Alexa Fluor 700 and CD27 BV480 from BD Biosciences (San Jose, CA); IL-21 PE from eBioscience, (San Diego, CA); and CD45RO PE-Cy5.5 from Beckman Coulter (Fullerton, CA). LIVE/DEAD Fixable Blue Dead Cell Stain Kit from Thermo Fisher Scientific (Boston, MA) was used to detect and exclude dead cells. All the reagents were tested and titrated for optimum concentration before usage.

Tumors were incubated with collagenase/DNase for 15 min, homogenized and first incubated with Fc Block™ (BD-Biosciences) for 10 min. Then, tumor samples were stained with the following antibodies to simultaneously quantify lymphocyte subsets, OVA(257-264) specific CD8 T-cells and expression of immune checkpoint molecules: CD45-APC-Cy7, K^b/OVA(257-264)-APC, CD4-Alexa 700, CD8-PE, CD3-BUV496, PD1-PerCP/Cy5.5, LAG3-BV650 and TIM3-BV785 (all from Biolegend, San Diego, CA, USA, except CD3-BUV496 which is from BD-Biosciences).
To analyze effector functions cells were stimulated for 4 h with PMA (100 ng/mL)/ionomycin (1 μg/mL) and then stained with antibodies against CD45-APC-Cy7, CD4-Alexa 700, CD8-BV421 CD107a-FITC (all from Biolegend), CD3-BUV496 (from BD-Biosciences). Then cells were fixed and permeabilized and finally stained intracellularly with anti-IFN-γ-PE and TNF-α-BV510.
In all cases the promofluor 840 (maleimide, Promokine, Burlingame, CA, USA) was added to stain dead cells. Samples were acquired with FACSCantoII (Becton Dickinson, Franklin Lakes, NJ, USA) or with Cytoflex (Beckman Coulter, Irving, TX, USA) flow cytometers and analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA).