Cd4 pe cy5

6-day stimulated, CFSE-labeled PBMCs were subjected to three different panels of staining to measure: phenotypic and cytokine profile, HIV infectivity (p24) and intracellular transcription factors (T-bet and EOMES) in CFSE-low, Ag-specific CD4 T cells. Briefly, cells were first stained for viability with LIVE/DEAD Fixable Aqua Blue (Life Technologies), followed by surface makers staining, including CD3-APC-H7 (BD Bioscience), CD4-PE-Cy5 (BD Bioscience), CD8-BV785 (Biolegend), CD25-PE-Cy5 (Biolegend), CCR5-Pacific Blue (BD Bioscience), α4β7-APC (NIH AIDS Reagent Program), CCR6-APC (R&D Systems), CCR9-PerCP-Cy5.5 (Biolegend). After surface staining, cells were fixed, permeabilized (BD Bioscience) and subjected to intracellular staining, including HIV p24-PE (Beckman Coulter), IL-17-Alexa Fluor 488 (eBioscience), IL-22-APC (eBioscience), IL-2-PerCP-Cy5.5 (Biolegend), IFN-γ-Alexa Fluor 700 (eBioscience), MIP-1β-PE-Cy7 (BD Bioscience). Combination of surface and intracellular staining antibodies may vary according to different assays. For intracellular staining for T-bet and EOMES, cells were fixed and permeabilized using eBioscience’s transcription factor staining buffer set (eBioscience) according to manufacturer’s instructions. Cells were then subjected to intracellular staining with anti-T-bet-Pacific Blue (Biolegend) and anti-EOMES PE-eFluor 610 (eBioscience).

ELISA kits for murine IL-6 was from R&D Systems (Minneapolis, MN). Fluorescein-conjugated mAbs including anti-CD3-PE-Cy7, CD4-PE-Cy5, CD8-PE, CD8-FITC, CD11b-APC, Ly6G-PE, Ly6C-FITC, CXCR2-Percp-Cy5.5, CD45-BV510, PD1-PE, PDL1-PE, LAG3-PE, CTLA4-PE, IFNγ-PE and isotype antibodies were purchased from BD biosciences. TIM3-PE was from Miltenyi Biotec (Bergisch-Gladbach, Germany). Antibody against STAT3 (79D7, 4904S), p-STAT3 (Tyr705, 9131S), ZEB1 (3396P), ZO-1(5406P), Snail(3879P), N-Cadherin(4061P) were obtained from Cell Signaling Technology (Beverly, MA, USA) and antibody against Actin was from Santa Cruz. IL6 inhibitor (501109) and IFN-γ inhibitor (505827) was from Biolegend. Luciferin substrate (K9909PE) for in vivo image was purchased from PerkinElmer Inc (Hopkinton, MA, USA). HE staining kit was from Beyotime Biotechnology. ShRNA for ZEB1 (TG513177) was purchased from OriGene.

Adult C57BL/6 mice were exposed to LPS (10 mg/kg) alone or combined with 4 g/kg ethanol for 16 h. Under CO₂ anesthesia, cardiac puncture was performed to obtain blood. After plasma and cell separation, the cell component was resuspended in Qiagen red blood cell lysis buffer, and the consequent white blood cells were isolated. Next, the cells were blocked with TruStain FcTMXPLUS (0.25 μg/ml; BioLegend), and subjected to immunostaining with the following surface marker antibodies: CD11b-FITC (5 μg/ml; Invitrogen), Ly6G-APC (4 μg/ml; BD Pharmingen), CD3e-Alexa 700 (10 μg/ml; BD Pharmingen), CD8-Pacific Blue (5 μg/ml; BioLegend), CD4-PE-Cy5 (10 μg/ml; BD Pharmingen), and CD19-PerCP Cy5.5 (10 μg/ml; BD Pharmingen). Next, the cells were permeabilized and fixed using the BD Cytofix/Cytoperm™ Fixation/Permeabilization Kit. Then, the cells were intracellularly stained with phospho-IκB-PE antibody (1.25 μg/ml; Invitrogen), followed by flow cytometry analysis.

Leukocytes were obtained from peripheral lymphoid organs (spleen, thymus, pancreatic and mesenteric lymph nodes) and blood. Red blood cells were cleared using 0.9% ammonium chloride solution. The leukocytes were then washed and spun down and were stained with antibodies for various leukocyte populations. The antibodies used were as follows: Fc block (anti-mouse FcRγIII/II mAb, 2.4G2, BD Biosciences, Franklin Lakes, NJ, USA), CD3-FITC (Clone: 17A2, BD Biosciences), CD4-PeCy5 (Clone: H129.19, BD Biosciences), CD8-APC-Cy7 (Clone: 53-6.7, BD Biosciences), CD19-PeCy7 (Clone: 1D3, BD Biosciences), CD25-APC-Cy7 (Clone: PC61, BD Biosciences), CD45 Pacific Blue (Clone: 30-F11, Biolegend, San Diego, CA, USA), and F4/80-APC (Clone: BM8, Invitrogen, Carlsbad, CA, USA). For intracellular FoxP3 staining, cells were incubated with FcRγIII/II, surface stained for CD4 and CD25, fixed and permeabilised as per manufacturer’s instructions (eBioscience, Waltham, MA, USA), and stained for FoxP3 (Clone: FJK-16S, eBioscience). Cells were then analysed using a FACSCanto flow cytometer (BD Biosciences) and Diva software (BD Biosciences). A total of 1 × 10⁶ cells were analysed.

BALF and lung cells were stimulated with M2e peptides (SLLTEVETPIRNEWGSRSN) (5 μg/mL) for 5 h at 37 °C in the presence of brefeldin A (BFA) (20 μg/mL). After stimulation, lymphocytes were stained with T cell marker mAb for CD4 (CD4-PE/Cy5, BD Biosciences) and CD8 (CD8α-PE, Biolegend) by following a procedure of BD Cytofix/Cytoperm Plus Kit. Intracellular staining of the permeabilized lymphocytes was conducted with IFN-γ cytokine mAb (anti-mouse IFN-γ-APC/Cy7, BD Biosciences). All samples were analyzed by using LSR-II/Fortessa flow cytometer (BD Biosciences, San Diego, CA, USA) and analyzed using the Flowjo software (FlowJo V10, Tree Star, Inc.).