Pma ionomycin

Primary T-cells were isolated from 3-4 mouse spleens by negative selection on magnetic bids using the Pan T cell Isolation Kit II (Miltenyi Biotech), see Grum-Schwensen et al. for details [13 (link)]. Purified T-cells were maintained in RPMI for 3 or 6 days with anti-CD3 or a combination of anti-CD3 and anti-CD28 antibodies, coupled to MACSibeads (Miltenyi Biotec) plus 10 ng/ml recombinant IL2 as described in [28 (link)].
Activated T-cells were stimulated with S100A4 protein (1 μg/ml) or S100A4 protein mixed with 6B12 antibody (6 μg/ml). Before fixation, PMA/Ionomycin and Golgistop™ (BD Biosciences) were added for 5 hours. Cells were washed with PBS and Fixable Viability Stain 450 (BD Biosciences) was added to discriminate between viable and dead cells.
Cells were fixed using the Cytofix/Cytoperm™ kit (BD Biosciences) and stained with the mouse Th1/Th2/Th17 phenotyping kit (BD Biosciences) containing antibodies against CD4, IL17A, IFNγ and IL4 according to the manufacturer’s instructions. Data acquisition and analysis were performed on a FACSVerse (BD Biosciences) using FlowJo software (Tree Star). All experiments were repeated 3-5 times.

All antibodies used for flow cytometry were purchased from BD Biosciences or eBiosciences and used according to the manufacturer's instructions. For neutrophil staining, Ly6G (FITC, 1A8), CD11c (APC-Cy7, N418), CD11b (PE Cy7, M170) and F4/80 (Percp Cy5, BM8) were used. For intracellular cytokine staining, CD3 (FITC, 145-2C11), CD4 (PercP, RM4-5), CD8 (PE Cy7, 53-6.7), IFN- γ (APC, XMG1.2) and TNF-α (PE, MP6X722) were used. For transcription factors, CD3 (FITC, 145-2C11), CD4 (APC Cy7, RM4-5), CD44 (PercP, IM7) and Tbet (Alexa 647, 4B10) or CD3 (PE, 145-2C11), CD4 (APC Cy7, RM4-5), CD44 (PercP, IM7), RorγT (APC, Q31-378) and Gata3 (Alexa 488, L50-823) were used. For memory cells, CD3 (PE, 145-2C11), CD4 (APC Cy7, RM4-5), CCR7 (Alexa 647, 3D12) and CD62L (FITC, MEL-14) were used. Briefly, tissue-isolated cells were incubated with monoclonal antibodies in buffer containing blocking antibody. For intracellular staining, the cells were harvested and plated with PMA-ionomycin and Brefeldin A (Golgi plug BD bioscience), and stained for flow cytometry analysis. After incubation, cells were fixed and permeabilized with the BD Cytofix/Cytoperm kit (BD Biosciences and eBiosciences, CA, USA). Cell acquisition was performed on the BD CANTO II cell analyser (BD Biosciences) using FACSDiva software (BD Biosciences), and data were analyzed using FlowJo software (Tree Star, Ashland, OR).

Agonistic anti-DR3 (4C12) monoclonal Ab and control Armenian Hamster IgG isotype (IgG) were purchased from BioLegend (San Diego, CA, USA). Anti-CD3e (2C11), anti-CD28 (37.51), anti-IL-17A (TC11-18H10), and CD16/CD32 (2.4G2) Abs were purchased from BD Biosciences (San Diego, CA, USA). T_regs were stained by using the FoxP3⁺T_reg staining kit following the manufacturer’s instructions (eBioscience, San Diego, CA, USA). Collagenase and DNase were obtained from Sigma-Aldrich (St. Louis, MO, USA), and dispase from Roche (Mannheim, Germany). RPMI-1640 cell culture medium (RPMI), fetal bovine serum (FBS), penicillin, and streptomycin (P/S) were all purchased from Invitrogen (Grand Island, NY, USA). Cytokines and other reagents were purchased from the following vendors: TGF-β1 and IL-6 (R&D Systems, Minneapolis, MN, USA), IL-2 (eBioscience), and PMA, ionomycin and GolgiStop (BD Biosciences). All ELISA kits were purchased from eBioscience.

Single cell suspensions were prepared and 1 × 10⁶ cells incubated in PBS + 0.1% BSA, 1% normal rat serum and appropriate antibody cocktails. Cell populations were determined and acquired on a BD FACS Fortessa (Becton Dickinson). Cell populations were identified by the following antibody staining strategies: CD4 T cells: CD3⁺CD4⁺; CD8 T cells: CD3⁺CD8⁺; B cells: CD19⁺B220⁺; Macrophages: CD11b⁺F4/80⁺; Dendritic cells: CD11c⁺. CD4 T cells populations were additionally stratified into naïve (CD44^loCD62L^hi) and activated (CD44^hiCD62L^lo) T cell populations and stained for Ox40 (CD134). Alternatively activated macrophages were characterised by staining for YM1 and RELMα. Intra-cellular cytokine staining was carried on MLN, spleen or lung cells. Cells were re-suspended in complete media (IMDM (GIBCO/Invitrogen; Carlsbad, CA), 10% FCS, P/S) at 2.5×10⁷/ml and stimulated with either10 μg/ml PMA/ionomycin or antigen and GolgiStop (as per manufacturer’s protocol; BD Pharmingen) at 37°C for 4 hours. After re-stimulation, cells were surface stained for CD3, CD4 then fixed and permeabilized with Cytofix/
Cytoperm Plus (as per manufacturer’s instructions; BD Pharmingen). Intracellular staining was performed by staining cells with either IL-13, IFN-γ or appropriately labeled isotype control. All analyses were performed with FlowJo software.