Tigit pedazzle594

Phenotypic markers expressed by CD4⁺ T cells isolated at day 0 and from day 14 were analysed by flow cytometry. Cells were fixed using Human FoxP3 Buffer Set (BD Biosciences, 560098), and stained with anti-human: CD4 BV510, LAG-3 Pe/Cy7, TIM-3 PE, TIGIT PeDazzle594, CTLA-4 BV421 (all BioLegend, 317444, 369310, 345006, 372716, 369606), CD3 FITC, and PD-1 APC (both ThermoFisher, 11-00390-42, 17-2799-42 respectively). Cells were then analysed using a cyAn ADP flow cytometer (Beckman Coulter).

Cell surface staining for flow cytometry was performed using the following antibodies: CD45-BUV395 (clone HI30, BD), CD3-AF700 (clone UCHT1, BD), CD4-APC-H7 (clone RPA-T4, BD), CD8-APC-H7 (clone SK1, BD), TIGIT-PE/Dazzle™ 594 (clone A15153G, Biolegend), TIGIT-BV421 (clone A15153G, Biolegend), and PD1-BV421 (clone EH12.2H7, Biolegend). These antibodies were used to analyze surface receptors in two different panels. Isotype-matched antibodies, labeled with the proper fluorochromes, were used as negative controls. Extracellular staining was performed according to the manufacturer’s instructions. Briefly, an antibody cocktail was added to whole blood or BM samples, which were then incubated at room temperature for 15 minutes in the dark, followed by red cell lysis with lysis buffer (BD; Cat: 555899). The lysed cells were washed twice with phosphate buffer saline (PBS), and then 350 μl stain buffer was added for further flow cytometer analysis. Twenty microliters of absolute count microsphere (Thermo; Cat: C36950) were added to the samples for absolute number analysis. Cells were analyzed using a BD Fortessa flow cytometer (BD Biosciences), and data analysis performed with Flowjo 10.6 software.