Fixation and permeabilization solution

The cells were preincubated for 20 min with anti-CD16/32 (2.4G2, Fc block from BioXCell) and stained for viability with LIVE/DEAD^® (Invitrogen, Carlsbad, CA) for 20 min on ice. Cells were surface stained for flow cytometry with the combination of the following antibodies (eBioscience or Biolegend): Anti-CD45 (104), anti-CD3 (145-2C11), anti-Thy1.2 (30-H12), anti-B220 (RA3-6B2), anti-Ter-119 (TER119), anti-Gr-1 (RB6-8C5), anti-CD11c (N418), anti-CD5 (53-7.3), anti-Eomes (Dan11mag), anti-T-bet (4B10), anti-RORγt (Q31-378, BD Pharmingen), anti-IFNγ (XMG1.2), anti-IL-17a (TC11-18H10.1), anti-Ly6G (1A8), anti-CD11b (M1/70), anti-CD64 (X54-5/7.1), anti-CD103 (2E7), anti-MHCII (M5/114.15.2). For intracellular cytokine staining was cells (1 x 10⁶ cells) were restimulated for 4 hours at 37°C with PMA (50 ng/ml) and ionomycin (500 nM) in the presence of brefeldin A (5 μg/ml) (all from Sigma-Aldrich, St. Louis MO) and fixed and permeabilized using fixation and permeabilization solution (eBiosciences). All cells were analyzed on either a FACS Celesta or LSR II (BD Biosciences) and analyzed with FlowJo software (FlowJo LLC).

Cells were obtained from bone marrow and spleen of FVB mice (Charles River Laboratories) after euthanasia under anesthesia. Cells were stained with rabbit anti-CLCF-1 polyclonal serum (Santa Cruz Biotechnology NNT-1/BSF-3 (FL-225)) which was raised against a human CLCF-1 peptide and cross-reacts with mouse and rat protein. Normal rabbit IgG (catalog #: sc2027) was used as an isotype control. Detection was performed with goat-anti-rabbit-FITC (Pharmingen 554020). Fixation and permeabilization solution from eBioscience was used as recommended. The data were analyzed using Becton-Dickenson FACSCalibur and FlowJo single cell analysis software.

The following monoclonal antibodies (eBiosciences) were used in appropriate combinations: anti-CD3 (clone 145–2C11), anti-CD4 (clone RM 4–5), anti-TCR γδ (clone GL3), anti-CD19 (clone 1D3), anti-CD11b (clone M1/70), anti-Gr1 (clone-RB6-8C5), eFlour 780 anti-CD90 (clone53-2.1) and anti-CD16/CD32 (clone 2.4G2). The cells were preincubated for 20 minutes with anti-CD16/CD32 to block Fc receptors to avoid nonspecific binding. Cells were then washed and labeled with the appropriate mixture of antibodies or isotype matched controls for 30 minutes, centrifuged at 650 g, and resuspended in flow cytometry staining buffer (FACS) buffer. To exclude dead/dying cells and therefore nonspecific antibody-binding cells, leukocytes were gated according to forward and side scatter. The percentages of CD4⁺, CD8⁺, CD4⁺ CD8⁺ and γδ T cell subsets were calculated on a CD19⁻ CD3⁺ gate. For intracellular cytokine staining, cells were restimulated for 4 h with 50 ng/ml PMA and 1 µg/ml Ionomycin (Sigma) and Golgi Stop and block (BD biosciences) were added for the last two hours. The cells were fixed and permeabilized using fixation and permeabilization solution (eBiosciences). Staining was performed for IL-4 (clone 11B11) and IFN-γ (clone XMG1.2) antibodies, and the cells were analyzed on a LSRII flow cytometer (BD Biosciences) using FlowJo software (Tree Star).