Tnf α fitc

Flow cytometry was performed using a FACSAria II cell counter (BD Biosciences). T cells were assessed for the surface presentation of epitopes with fluorescently labeled monoclonal antibodies for CD69 (BioLegend). In parallel, anti‐CD19 CAR expression was measured using Flow cytometry. First, anti‐CD19 CAR expression was measured with commercial bio‐sCD19‐Fc (ACROBiosystems), followed by staining with secondary APC‐conjugated SA antibody (BioLegend). Anti‐CD19 CAR expression was measured with sCD19‐SA, followed by a secondary PE‐conjugated anti‐SA antibody (BioLegend), and anti‐CD19 CAR expression was directly measured with FITC‐sCD19‐SA. To assess cytokines produced by CAR‐T cells, cells were incubated in 96‐well U‐bottom plates at 10⁵ cells/100 μl media/well for 24 h in the presence of 60 μg/ml sCD19‐SA with 5 μg/ml Brefeldin A (BioLegend), and the hGM‐CSF‐SA fusion protein (prepared in our laboratory) was added to the medium alone as a negative control. Before permeabilization with ice‐cold methanol, the cells were fixed in 1.5% formaldehyde and then incubated with antibodies against FITC‐TNF‐α, PE‐IFN‐γ, and APC‐IL‐2 (all from BioLegend) for flow cytometric analysis. A total of 10⁴ cells were counted by flow cytometry for each experiment.

Nr-CWS was supplied by Greatest Biopharma Limited Company (purity>98%). Fluorescein conjugated anti-mouse antibodies APC-NK1.1, FITC-NK1.1, APC/Cy7-CD3ε, PE/Cy7-CD3ε, APC-CD4, PE-CD8α, PerCP/Cy5.5-CD11b, PE-CD27, FITC-TNF-α, and PerCP/Cy5.5-IFN-γ from BioLegend, and PerCP/Cy5.5-CD69, FITC-FasL, PE-TRAIL, PE-Perforin, and FITC-Granzyme-B from eBioscience were used in this study. TUNEL assay kit and MTS Cell proliferation, cytotoxicity or chemosensitivity assays kit (cat # 3580) were obtained from Promega. The LEGENDplex™ Multiplex mouse cytokine kit was provided by BioLegend. EasySep™ mouse NK cell isolation kit was from STEMCELL Technologies Inc. Recombinant murine IL-2 was from PeproTech Inc.

Cells were washed once in FACS buffer (PBS containing 0.1% BSA) before blocking with anti-FcγRII/FcγRIII (2.4G2, BD Pharmingen). Staining of surface molecules (all Biolegend) was performed on ice using FITC-conjugated CD4 (RM4-5), CD8α (53-6.7), and CD90.1 (OX-7); PECy7-conjugated CD44 (IM7); PE-conjugated α4β7 (LPAM-1, DATK32), CD4 (RM4-5), CD8α (53-6.7), CD62L (MEL-14), CD44 and CD25 (PC61); APC conjugated CD90.1 (OX-7). Intracellular Foxp3 (FJK-16s, APC-conjugated; eBioscience), NFATc1 (anti-NFATc1 PE 7A6, Biolegend), IRF4 (3E4, APC or PB-conjugated; Biolegend) staining was performed using the Foxp3 staining kit (eBioscience) according to the manufacturer’s instructions. Antibodies (all Biolegend) for intracellular cytokine staining were APC-IFN-γ (XMG1.2), FITC-TNF-α (MP6-XT22) and PE-IL-2 (JES6-5H4). Cytokine detection was performed after a 6 h in vitro restimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA; 10 ng/mL, Sigma) plus ionomycin (5 nM, Merck Biosciences) in the presence of GolgiStop and GolgiPlug (both BD Pharmingen) using the IC Fixation Buffer kit (eBioscience). Viable cells were detected with the Zombie Aqua™ Fixable Viability Kit (Biolegend). Data were acquired on a FACSCanto II (BD Biosciences) flow cytometer and analyzed with FlowJo software (Tree Star).