Gilgfvftl

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.

PBMCs or Adherence DC preparations from A0201 donors were co-cultured with apoptotic 3T3 (DC/ctrl) or apoptotic influenza-infected 3T3 cells (DC/flu) for 5 hours in GolgiStop (BD Bioscience) before staining with APC-conjugated A0201 Influenza-M1 dextramer (GILGFVFTL, Immudex) followed by CD8-FITC (BD Bioscience). Cells were fixed and permeabilized (Cytofix/Cytoperm, BD Bioscience) as per manufacturer’s protocol. Intracellular cytokine staining was performed using IFNγ − PE (BD Bioscience) followed by flow cytometry analysis.