Cd95 pe cy5

The procedure of flow cytometric analysis of CMV- and IAV-specific CD8⁺ T cells prior to and post in vitro stimulation was described [40 (link)]. Briefly, freshly PMBCs were stained with fluorescently labeled dextramers or tetramers specific to CMV-pp65 (NLVPMVATV) and IAV-M1 (GILGFVFTL) (Immudex, Copenhagen, Denmark and NIAID tetramer core) first at room temperature for 20 min; followed by staining of cell surface markers including antibody against CD3, CD8, CD62L, CD45RA, CD95, CD27, CD28 CD70, CD127, and CD69 at 4°C for 30 min. Cells were washed again with FACS buffer (Hanks solution with 0.3% Sodium Azide), and then fixed immediately with 3% formaldehyde and 1% FBS in FACS buffer. Fixed cells were further stained with intracellular markers (perforin and granzyme B) at 4°C for 30 min. Stained cells were collected by BD_Symphony and were analyzed using FlowJo version 7.6.5 software.
Antibodies (CD3-BV570, CD62L-FITC, CD62L-PE-Cy7, CD45RA-PE-Cy7, CD45RA-APC, CD95-PE-Cy5, CD27-PE, CD28-BV785, CD28-PerCP-Cy5.5, CD70-PE, CD127--BV711, CD69-BV650, and granzyme B) were purchased from Biolegend, and antibodies against CD8-BUV496, CD27-BUV395, and perforin-PE-CF594 were purchased from BD.

Thawed PBMCs were reacted with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific, Waltham, MA, USA) to remove the dead cells. For phenotypic analysis, cells were stained with phycoerythrin (PE)-conjugated Tax_301-309/HLA-A*24:02 tetramer reagents (MBL, Nagoya, Japan) and several fluorescence-conjugate mouse anti-human monoclonal antibodies (mAbs) [CD3-APC-H7, CD8-Pacific Blue, CD45RA-PerCP5.5, CCR7-Alexa647, CD62L-PE-Cy7, CD27-FITC, CXCR3-BV605 (BD Biosciences), and CD95-PE-Cy5 (Biolegend, San Jose, CA)] for 25 min on ice. Stained cells were washed twice and immediately acquired using FACSAriaIII Fusion (BD Biosciences) equipped with 20 detectors by 4-lasers at 488 nm, 561 nm, 633 nm, and 405 nm. The data were analyzed using FlowJo ver.10 software (BD Biosciences). The experiments requiring cell sorting for TCR repertoire analysis, described below, were carried out using the same equipment.