Fixation and permeabilization solution

To determine immunophenotyping of Th9, Th17, Th22, and Treg cells, surface staining of CD4 and CD25 markers was performed using mouse anti-human CD25-Alexa Fluor 488 (eBioscience, San Diego, CA, USA), CD4-PE (Immunostep, Salamanca, Spain), and CD4-FITC (BioLegend, San Diego, CA, USA) for 30 min at 4°C. Intracellular staining was followed by washing the cells and incubation with 1× fixation and permeabilization solution (BD Biosciences, San Diego, CA, USA) for 15 min at 4°C. The cells were subsequently washed with cold PBS, and intracellular staining was performed by mouse anti-human IL-17A-Alexa Fluor 660 (eBioscience, San Diego, CA, USA), mouse anti-human IL-9-PE (BioLegend, San Diego, CA, USA), mouse anti-human IL-22-PE (BioLegend, San Diego, CA, USA, Cat. No. 366703), and anti-human FOXP3-APC (eBioscience, San Diego, CA, USA) for 30 min at 4°C and kept in the dark. IgG1 Isotype-matched antibody controls were used for all staining. We used anti-human CD4-FITC for evaluation of CD4+IL-22+ and CD4+IL-9+ T cells, and anti-human CD4-PE for evaluation of Treg cells. Ten thousand events were evaluated with BD FACSCalibur (BD Biosciences) and analyzed using FlowJo Software version 10 (BD Company, USA).

LMCs (1 × 10⁵) were cultured on a 6-cm cell culture dish overnight and were treated with TNFα (10 ng/ml) for 0.5, 1, 2, or 10 h. Cells were harvested by trypsin digestion and were fixed with a Fixation and Permeabilization Solution (BD Biosciences, #554722) according to the manufacturer’s instructions. Cells were stained with the rabbit antibody against pERK1/2 (Cell Signal Tech, #4695) followed by the anti-rabbit Alexa 488 antibody (Cell Signal Tech, #4412S). Flow cytometry (BD, LSR Fortessa SORP) was performed to detect samples, and data were analyzed using the software FlowJo (BD, V10).

The cells were preserved and permeabilized for 20 min at 4°C utilizing fixation and permeabilization solution (BD Bioscience, Franklin Lakes, NJ, USA). Following two rinses with perm/wash buffer (BD Bioscience, Franklin Lakes, NJ, USA), the cell pellet was resuspended and blocked with 2% bovine serum albumin (BSA) at room temperature for 15 min, then incubated for 1 h at 4°C with the suggested quantity of antibody (CD146, GABARAPL1, or NLRP3). Before conducting the flow cytometric examination, the cells were treated with fluorochrome-conjugated secondary antibodies, then incubated for 30 min at 4°C in the dark and rinsed two times.

2x10⁶ splenocytes from each sample were plated in a 96-well plate. After centrifugation, cells were resuspended and incubated in culture medium (R10) consisting of RPMI 1640 containing 10% FBS (Mediatech, Herndon, VA), 2mM L-glutamine, 0.01 M HEPES buffer, 100mg/ml gentamicin (Mediatech), and 5×10^-5M 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO). To test intracellular cytokine, cells were stimulated with 30 mg/ml PMA and 400 ng/ml ionomycin in the presence of GolgiStop (BD Pharmingen) for 4 hours at 37°C.
After incubation and stimulation, cells were surface-stained with anti-CD3-PB (BD), anti-CD4-PerCP (BD), anti-CD8-PO (Biolegend), anti-BTLA-PE, anti-2B4-APC, anti-PD-1-APC-Cy7 (all from eBioscience). Then cells were permeabilized using fixation and permeabilization solution (BD). We used anti-IL-2-FITC (BD), anti-TNF-PE-Cy7 (Biolegend) and anti-IFN-γ-Alexa 700 (BD) for intracellular cytokine staining. In some experiments, cells were stained intracellularly for Ki-67 using the Foxp3 staining kit (BD Pharmingen). Samples were analyzed on an LSRII flow cytometer (BD) and data were analyzed using FlowJo software (FlowJo, LLC) and SPADE (Cytobank.org) (see below).

1.5 × 10⁶ cells were resuspended in complete RPMI 1640 medium, then stimulated with phorbol 12-myristate 13-acetate (PMA) (20 ng/ml, Sigma) and ionomycin (1 µg/ml, Sigma) for 1 h. Brefeldin A (BFA, 10 µg/ml, Sigma) was added and incubated for 4 h. Cells were washed twice in PBS and stained with specific antibodies for the cell surface antigens for 30 min at 4 °C in the dark. Cells were fixed with Fixation and Permeabilization Solution (BD Biosciences) for 20 min at 4 °C in the dark. Next, cells were stained with specific antibodies for each cytokine. The results were analysed using flow cytometry (Beckman CytoFLEX) and CytExpert 1.1 (Beckman Coulter Inc.). The single nuclear cells were gated to exclude the dead cells and doublet. For gating CD3⁺ γδTCR⁺ cells, CD3⁺, CD3⁺ CD4⁺, CD3⁺ CD8⁺, CD3⁻ CD19⁺ cells, FMO controls were used. Isotype controls were used for intracellular cytokines staining. 1,500,000 cells were used for cell intracellular cytokine staining, and 300,000 events were collected for each tube.

For all staining, cells were blocked with 0.1% BSA prior to extracellular staining, then, if intracellularly stained, cells were treated with Fixation and Permeabilization Solution (BD) and stained for intracellular markers in Perm/Wash Buffer (BD). Flow cytometry was performed on an LSR II and analyzed in FlowJo software, both courtesy of the University of Wisconsin Flow Core facility. Stains: Hoechst, and antibodies against PD-L1 (BD, MIH1), MUC1 (Biolegend, 16A), CD11b (Biolegend M1/70), CD34 (Biolegend, 581), CD45 (Biolegend, H130; Tonbo, HI30; or abcam GA90), pan-Cytokeratin (pCK, abcam, C-11), and isotype control, IgG1 (abcam, CT6).