Facscalibur machine

To obtain single cell suspensions, between 40 and 50 anesthetized mutant and wild-type 48hpf embryos were incubated for 20 minutes on a shaker in 0.25% trypsin in L15 tissue culture media (Sigma). Repeated trituration using fire-polished glass pipettes was performed. Cell suspensions were cleaned with a mesh and re-suspended in PBS. Cells were fixed in 70% EtOH and stored at 4ºC for several days. Cells were re-suspended in 100 microliters of propidium iodide solution (in 4 mM citrate buffer, pH 6.5, containing 0.1 mg/ml propidium iodide (Sigma), 200 μg/ml RNase, and 0.1% Triton X-100) and stored at 4ºC until analysis. Data acquisition was performed by using a Becton Dickinson FACS-Calibur machine and analyzed by using the FlowJO programme.

Cell wall display of mouse IL-22 on the surface of Lactobacillus was confirmed by flow cytometric analysis. Fifty μl of Lactobacillus (Lp pAFβ900-IL22) cultures grown in MRS to an OD₆₀₀ of 1.0 were pelleted by centrifugation (3000×g for 10 min) and washed twice in PBS. Bacteria were incubated with biotinylated goat anti-mouse IL-22 antibody (1 μg/ml) for 30 min on ice. After two PBS washes, the cells were incubated with FITC conjugated streptavidin (BioLegend, San Diego, CA, 1:500 dilution) on ice for 30 min in the dark. The antibody and conjugated streptavidin were diluted in PBS containing 1% BSA. After washing three times with PBS, samples were resuspended and fixed in 400 μl 1% paraformaldehyde and analyzed using a FACS Calibur machine (Becton–Dickinson, Franklin Lakes, NJ).

Cells from spleens and lymph nodes were depleted of erythrocytes by hypotonic lysis. The cells were washed with FACS washing buffer (2% FBS, 0.1% NaN₃ in PBS) twice and were then incubated with fluorescence-conjugated antibodies against cell surface molecules for 30 min on ice in the presence of 2.4G2 mAb to block FcγR binding. Isotype antibodies were included as negative controls. For intracellular cytokine staining, single-cell suspensions were stimulated with 50 ng/ml PMA and 1 μM ionomycin in the presence of brefeldin A solution for 4 h. After stimulation, cells were stained with fluorescence-conjugated antibodies against CD4 and CD25, fixed and permeabilised using a fixation/permeabilisation kit (eBioscience, San Diego, CA, USA) and stained with fluorescence-conjugated specific antibodies against IFN-γ, IL-17A, and Foxp3 in accordance with the manufacturer’s instructions. Flow cytometry was performed using a Becton Dickinson FACSCalibur machine.

Fluorescent-dye-labeled antibodies against cell-surface markers CD4, CD8, CD44, CD62L, Gr1, CD19, CD45.1, CD45.2, CD127, CD27, and KLRG1 were purchased from eBioscience (San Diego, CA, USA). FITC- or PE-conjugated antibodies against cytokines were from BioLegend (San Diego, CA, USA). APC- or PE-conjugated K^b-ova+ tetramer was obtained from QuantoBio (Beijing, China). Splenic cells were depleted of erythrocytes by hypotonic lysis. The cells were washed with FACS washing buffer (2% FBS, 0.1% NaN₃ in PBS) twice and were then incubated with fluorescence-conjugated antibodies against cell-surface molecules for 30 min on ice in the presence of 2.4G2 mAb to block FcγR binding. Isotype antibodies were included as negative controls. For intracellular cytokine staining, single-cell suspensions were stimulated with 10 nM SIINFEKL peptide in the presence of Brefeldin A solution (eBioscience) for 6 h at 37 °C. After stimulation, cells were stained with fluorescence-conjugated antibodies against cell-surface markers, fixed, and permeabilized using a fixation/permeabilization kit (eBioscience) and stained with fluorescence-conjugated specific antibodies against Smad4 (Santa Cruz, Santa Cruz, CA, USA), IFN-γ, TNF-α, and GzmB in accordance with the manufacturer's instructions. Flow cytometry was performed using a Becton Dickinson FACSCalibur machine.

The pool of rat articular chondrocytes was seeded at a density of 20.000 cells/cm² in T-25 cell culture flasks (passage 4). After 24 hours the cells were either treated with dPGS-ICC or as a control with glycerol-ICC (both groups: 10⁻⁶ mol/L, mivenion GmbH, Germany, for 2 hours, 72 hours and 7 days). One flask remained untreated as control. Then, trypsinized cells were fixed with 4 % PFA and resuspended in FACS buffer. 10.000 events per sample have been counted using a FACSCalibur machine (Becton, Dickinson and Company, USA) and data was analyzed using the FlowJo Software (version 7.6, Tree Star, Inc., USA).

10⁷ epimastigotes from a dense culture were centrifuged and resuspended in 100 µL of TbBSF buffer [36 (link)] containing 10 µg of circular (for transient transfection) or linearized (for stable transfection) plasmid DNA. The plasmid pTcRG was kindly provided by Santuza Teixeira (Federal University of Minas Gerais, Belo Horizonte, Brazil). The cells were electroporated with a nucleofector device (Lonza) in a 0.2 mm cuvette (BioRad, Hercules, CA, USA). After electroporation, the cells were transferred to 10 mL of LIT with a fine-tipped Pasteur pipette. The parasites transfected with circular plasmids were incubated for 24 h and then tested for GFP expression with flow cytometry with a FACSCalibur machine (Becton Dickinson and Company, Franklin Lakes, NJ, USA). The parasites transfected with linearized plasmid were incubated for 24 h, diluted 1:10 in medium containing 100 µg/mL G418 (Gibco, Billings, MT, USA), and further distributed in a fourfold dilution series in a 48-well plate under antibiotic pressure. Outgrowing epimastigotes were cloned by limiting dilution and assessed for correct integration of the transgene with PCR and Southern blot.