Ifn γ pe cy7 clone b27

The following conjugated Abs were used for cell surface staining: CD3-Pacific Blue (clone UCHT1), CD4-Alexa Fluor 700 (clone RPA-T4), CD8-APC-H7 (clone SK1), CD160-Alexa Fluor 647 (clone BY55), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3)-PE-CF594 (clone 7D3), 2B4-FITC (clone 2–69) (BD Biosciences, San Jose, CA), and programmed cell death protein-1 (PD-1)-PE (clone J105) (Thermo Fisher Scientific). Conjugated Abs for intracellular staining included the following: cytotoxic T lymphocyte antigen-4 (CTLA-4)-PE-Cy5 (clone BNI3), granzyme B-PE-CF594 (clone GB11), interferon-gamma (IFN-γ)-PE-Cy7 (clone B27), interleukin-2 (IL-2)-APC (clone MQ1-17H12), tumor necrosis factor-alpha (TNF-α)-Alexa Fluor 488 (clone MAb11) (BD Biosciences, San Diego, CA), and perforin-PE (clone B-D48) (Abcam, Cambridge, UK). All conjugated Abs were titrated and multicolor panels for flow cytometry assays were approached as previously reported [38 (link)]. The following purified (No azide/Low endotoxin) Abs were used in cultured cells: CD28 (clone CD28.2) and CD49d (clone 9F10) (BD Biosciences).

Cells were collected after 72 h of recall stimulation and treated with 5 µg/ml of Brefeldin A and 5 µg/ml of Monensin for 4 h. Then, cells were centrifuged for 3 min at 500 × g and supernatants were removed. Cells were stained with Aqua Viability Dye (Molecular Probes/Invitrogen) for 20 min in RT and thereafter labeled with conjugated monoclonal antibodies (mAbs) against cell surface markers for 30 min at 4°C. After wash and treatment with perm/wash buffer (BD Biosciences) for 20 min at 4°C, the cells were stained for intracellular cytokines for 30 min at 4°C. The following mAb conjugates were used (BD Biosciences): CD3-APCH7 (clone SK7), CD4-FITC (clone RPA-T4), CD8-PerCPCy5.5 (clone SK1), CD45RO-APC (clone UCHL-1), CCR7-PECF594 (clone 2-L1-A), CD28-PE (clone CD28.2), CD95-BUV395 (clone DX2), IFNγ-PeCY7 (clone B27), MIP-1β-AF700 (clone D21-1351), IL2-BV711 (clone 5344.111), and TNF-BV421 (clone MAb11). CD14-V500 (clone M5E2) and CD19-V500 (clone H1B19) were included in the dump channel. PBMCs were acquired using a ZE5 flow cytometer (Bio-Rad) with Everest software (Bio-Rad). Results were analyzed using FlowJo software (BD Biosciences).