Fitc conjugated anti ifn γ

The functions of NKT-like cells in PBMCs were detected after coincubation with the K562 cell line or PMA/ionomycin. Because NKT-like cells share some features with NK cells, they can recognize HLA class I-negative cell line K562 and their functions can be detected through coculture with K562 [10 , 11 (link)]. PBMCs were coincubated for six hours with K562 cell line at an E : T ratio of 5 : 1. Meanwhile, PBMCs were stimulated with PMA (Sigma, Cat. No. P-8139, USA) and ionomycin (Sigma, Cat. No. I-0634, USA) in final concentrations of 50 ng/mL and 1 μM, respectively. PE-conjugated anti-CD107a (BD Biosciences, USA) and monensin (Becton-Dickinson, USA) were added to all incubated samples. Then cells were stained with both Percp-conjugated anti-CD3 and PE-cy7-conjugated anti-CD56 (BD Biosciences, USA). The cells were made permeable using Perm/Wash (Becton-Dickinson, USA) for 10 minutes, stained with FITC-conjugated anti-IFN-γ (BD Biosciences, USA) for 30 minutes at 4°C, washed, and then fixed in 1% formaldehyde. NKT-like cell populations were defined by dual-positive expressions of CD3 and CD56 molecules. The frequency of IFN-γ and CD107a expression in NKT-like cells were quantified by multicolor flow cytometry (Figure 1).

Surface and intracellular phenotyping of T cells was performed by flow cytometry as previously described [9 (link), 18 (link)]. Allophycocyanin-conjugated anti-CD3, allophycocyanin- or phycoerythrin (PE)-conjugated anti-CD4, peridinin chlorophyll protein-conjugated anti-CD8, fluorescein isothiocyanate (FITC)-conjugated anti-CD95, PE-conjugated anti-CD25, PE-conjugated anti-HLA-DR, FITC-conjugated anti-IFN-γ, PE-conjugated anti-IL-4 (BD Biosciences, San Jose, CA), FITC-conjugated anti-IL-17A (eBioscience, San Diego, CA), anti-ERα (clone C-542, Abcam, Cambridge, UK), and anti-ERβ (clone 1531, Santa Cruz Biotechnology, Santa Cruz, CA) monoclonal (m)Abs were used. Anti-ER Abs were visualized by FITC-conjugated F(ab’)2 fragment secondary Ab (Abcam). Equal amount of mouse IgG isotype controls were run in parallel. To determine the frequency of T cell subsets, total lymphocytes were first gated by forward and side scatter and then additionally gated for CD4 or CD8 molecule expression. Acquisition was performed on a FACSCalibur flow cytometer (BD Biosciences) and 50,000 events per sample were run. Data were analyzed using the Cell Quest Pro software (BD Biosciences).

Cells were pre-incubated with PMA (5 ng/ml) and ionomycin (500 ng/ml) for 6 h, and with brefeldin A for 5 h (10 μg/ml; all from Sigma-Aldrich). For cytokine detection, FITC-conjugated anti–IFN-γ (Clone: B27, Cat# 554700, RRID : AB_395517), PE-conjugated anti-IL-5 (Clone: JES1-39D10, Cat# 559332, RRID : AB_397229), or IL-17 (Clone: SCPL1362, Cat# 560436, RRID : AB_1645514), BV711 conjugated anti-IL-13 (Clone: JES10-5A2, Cat# 564288, RRID : AB_2738731), and allophycocyanin-conjugated anti-IL-4 (Clone: 8D4-8, Cat# 560671, RRID : AB_1727546) or IL-21 (Clone: 3A3-N2.1, Cat# 562043, RRID : AB_10896655, all from BD Biosciences) were used alone or together. Isotype control Abs included FITC-conjugated mouse IgG1 κ (Clone: MOPC-21, Cat# 551954, RRID : AB_394297), PE-conjugated rat IgG2a κ (Clone: R35-95, Cat# 559317, RRID : AB_10050484), BV711-conjugated rat IgG1 κ (Clone: R3-34, Cat# 563283, RRID : AB_2869482), and Alexa Fluor (AF)-647-conjugated mouse IgG1 κ (Clone: MOPC-21, Cat# 557732, RRID : AB_396840, all from BD Biosciences).

PBMCs and SFMCs were isolated and suspended in a complete medium (RPMI 1640, 2 mM L-glutamine, 100 units/ml of penicillin, and 100 μg/ml of streptomycin) supplemented with 10% fetal bovine serum (FBS; Gibco BRL, Grand Island, NY, USA), and then seeded into 96-well plates at cell density of 1 × 10⁶ cells/well. Cells in a 96-well culture plate were treated with CSp and then were activated with Dynabeads Human T-Activator CD3/CD28 (11131D, Gibco, USA) to obtain a bead to cell ratio of 1:1. Cells were then incubated in a humidified CO₂ incubator at 37°C for 24 h. After stimulating with PMA (100 ng/ml) and ionomycin (1 μM) for 4 h, cells were stained with Pacific Blue-conjugated anti-CD4 (300521, BioLegend, USA), and PE-conjugated anti-CD45RO (304205, BioLegend, USA). Cells were washed, fixed, permeabilized with Cytofix/Cytoperm buffer, and stained intracellularly with FITC-conjugated anti-IFN-γ (552887, BD, USA), APC-conjugated anti-IL-17A (512334, BioLegend, USA) antibodies followed by analysis with FlowJo Software (BD, USA). In ex vivo cultured supernatants from PBMCs, INF-γ, IL-17A, TNF-α, and IL-6 were measured using ELISA (88-7316, 88-7176, 88-7346, and 88-7066, Invitrogen, Austria). The OD was recorded by a SpectraMax^® M2(Molecular Devices Corp., USA) set at 450 nm.

A flow cytometry-based analysis of TIL function was designed to detect CD107a membrane expression and intracellular IFN-γ expression. Briefly, TIL were stimulated at 37 °C overnight at a concentration of 5 × 10⁵ cells/mL in the presence of 25 ng/mL phorbol myristate acetate (PMA) and 0.5 μM ionomycin. After 90 min of stimulation, APC conjugated CD107a or its IgG1 k isotype (BD Biosciences) were added. Brefeldin A, at 1 μg/mL, (Sigma Aldrich) and monensin, at 0.067 % (v/v), (GolgiStop, BD Biosciences) were added one hour later. After 18 h, cells were washed once with Staining Buffer (PBS containing 0.05 % (v/v) Human AB serum), then surface stained (4 °C for 30 min.) with AlexaFluor 700, PECy7, and PerCpCy5.5 conjugated anti-CD3, CD4, and CD8 (BD Biosciences), respectively. The LIVE/DEAD Aqua (Invitrogen) dye was included as a cell viability stain. Cells were washed once with Staining Buffer and then fixed and permeabilized with BD Cytofix/Cytoperm for 20 min at 4 °C and washed twice with BD Perm/Wash (BD Biosciences). Intracellular staining of FITC conjugated anti-IFN-γ (BD Biosciences) occurred at 4 °C for 30 min, followed by one wash with BD Perm/Wash. Cells were stored in 0.5 % paraformaldehyde (Thermo Fisher Scientific) at 4 °C in the dark. All samples were run on an LSRII flow cytometer (BD Biosciences) within 48 h and analyzed using FlowJo software (TreeStar, Inc.)