Lsrii sorp cytometer

Chondrocytes from Cse KO, Cse tg, Cbs KO and corresponding WT mice were resuspended in fluorescence-activated cell sorting (FACS) buffer (5% FCS, 5 mM EDTA in PBS). For each condition, 10⁶ cells were incubated with the H₂S fluorescent probe P3 (10 µM) [42 (link)] and after two minutes FACS analysis was performed on a LSRII SORP cytometer (BD Biosciences, Franklin Lakes, NJ, USA) with a UV laser. FACS Diva (BD Biosciences) and FlowJoX (Tree Star, Ashland, OR, USA) software were used for data processing.

Panel 1 included antibodies allowing the gating of PMN-MDSCs in the PBMCs and the analysis of expression of surface markers. It comprised of CD3-BV510, CD56-BV510, CD19-BV510, CD11b-APCH7, CD15-BV711, CD14-BV650, HLA-DR-BV605, CD10-PE-CF594 (Becton Dickinson), CD16-AF700 (BioLegend, Ozyme, Saint-Cyr-l’Ecole, France), CD33-PC7 (eBiosciences, Thermo Fisher), LIVE/DEAD fixable aqua (Life Technologies, Villebon-sur-Yvette, France). LOX-1-PE, CD11b-activated-PE (clone CBRM1/5) (Bio-Legend, Ozyme), CD62L-PE, CD66b-PE, PDL-1-BV421, CXCR1-PE, CD172ab-AF647 (BD Biosciences) were alternately included in Panel 1.
Panel 2 was used for functional analysis. It contained CD15-FITC (eBiosciences, Thermo Fisher), CD3-PC5 (BD Biosciences) and LIVE/DEAD near-IR (Live Technologies,). CD66b-PE, CD10-PE-CF594 and CD16-AF700 were occasionally added. PBMCs were stained 20 min at room temperature with fluorescent reagents pre-mixed in PBS, washed, then fixed with 4% paraformaldehyde and analyzed by FACS within 4 days on LSRII-SORP cytometer (BD Biosciences) equipped with four lasers (405 nm/100 mW, 488 nm/100 mW, 560 nm/50 mW and 630 nm/40 mW). PMT were set using unstained and fully stained samples. Compensations were performed with beads stained with corresponding reagents. Data were exported and analyzed with FlowJo (version 9-2, MacOS X).

PBMC were stained with multiparametric panels containing, respectively, 9 and 12 fluorescent markers designed to investigate NK and DC populations (Supplementary Methods). Cells were incubated for 20 min at room temperature (RT) with reagents pre-mixed in phosphate buffered saline (PBS), washed, then fixed with 4% paraformaldehyde (PFA). Data were acquired on LSRII-SORP cytometer (BD Biosciences, Le Pont-de-Claix, France) equipped with four lasers (405 nm/100 mW, 488 nm/100 mW, 560 nm/50 mW and 630 nm/40 mW). Photo-multiplicator (PMT) were set using unstained and fully stained samples. Events of 7.74 × 10⁶±3.10⁵ were recorded. Compensations were performed with beads stained with corresponding reagents. Data were exported and analyzed with FlowJo (FlowJo LLC, Ashland, OR, USA) (version 9-2, MacOS X). NK cells were gated in the CD3⁻CD14⁻CD19⁻ gate as CD56⁺CD16⁺, or CD56⁺CD16⁻, or CD56⁻CD16⁺ cells. DC’s were defined as CD3⁻CD14⁻CD19⁻CD56⁻CD16⁻HLA-DR⁺ cells. For computation, a pre-analysis was performed on all samples (Supplementary Figure S1). The remaining data (3.5 × 10⁵±2.5 × 10⁴ events) were exported to create new files further subjected to automated gating (Supplementary Figure S1 and below).