Caspase 3 7 green reagent

OPCs were stained with an eFluor 633 dye prior to the addition of CD8s to ensure subsequent quantification was restricted to the OPC population. Unstained OT-1 CD8s were isolated and added to the stimulated and stained OPCs. Cell membrane permeable, caspase 3/7 Green reagent (1:1000; Essen Biosciences) was added to determine caspase activity. The caspase 3/7 reported was designed to report on caspase 3/7 activity by targeted cleavage of the DEVD peptide sequence and subsequent nuclear translocation of the linked DNA binding dye (Essen Biosciences). Images were captured every 2–3 h using IncuCyte S3 Live Cell Analysis System (Sartorius). Cultures were analyzed for caspase 3/7 stain colocalization with NucLight Rapid Red (Essen Biosciences) labeled OPCs and the applied mask was counted as particles per image with 9 images taken. A total of three experiments were completed. MHC class I inhibitors were applied two hours before antigen was given to IFNγ stimulated OPCs and kept in culture for the antigen loading portion of the experiment until CD8s were applied and antigen, IFNγ and inhibitors were washed away from the culture. Fas/FasL and Granzyme B inhibitors were applied to the culture at the time of OPC-CD8 co-culture initiation and were kept in culture for the remainder of the experiment.

Caco2 cells were seeded in a 96-well plate at a density of 2.5 × 10³ cells/well. Cells were grown overnight to 25–30% confluence at the start of the assay. Media were supplemented with 5 μM of Caspase-3/7 (green) reagent (4440, Essen Bioscience, Morgan Rd, Ann Arbor, MI, USA) or Annexin V (red) reagent (4641, Essen Bioscience, Morgan Rd, Ann Arbor, MI, USA) diluted to 1:200 and added to the cells treated with 0.01% of DMSO, different concentrations of B, and EL000327 for 48 h in the presence or absence of Z-VAD-FMK (10 µM). The apoptosis of cells was determined by fluorescence scanning performed every 2 h for 48 h by the IncuCyte Zoom^® instrument with a 10× objective and analyzed with the Standard Scan Type.

DMEM medium, DMEM red-phenol-free medium, RPMI 1640 medium, RPMI 1640 red-phenol-free medium, fetal bovine serum (FBS), L-glutamine and penicillin-streptomycin were purchased from Gibco BRL (Cergy-Pontoise, France). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), N-acetyl-L-cysteine (NAC), 3-methyladenine (3-MA), anti-β-actin antibody, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide (JC-1) and cell death detection enzyme-linked immunosorbent assay^PLUS (ELISA) were obtained from Sigma-Aldrich (Saint-Quentin-Fallavier, France). Beclin-1, Atg5 and LC3 antibodies were acquired from Cell Signaling Technology—Ozyme (Saint-Quentin-en-Yvelines). 2’,7’-dichlorofluorescein diacetate (DCFDA) cellular ROS detection assay kit and goat anti-rabbit IgG H&L horseradish peroxidase (HRP) secondary antibody were purchased from Abcam (Paris, France). LysoTracker, MitoTracker, rabbit anti-mouse IgG-IgM H&L HRP secondary antibody, TO-PRO-3, annexin V-FITC and propidium iodide (PI) were obtained from Invitrogen—Thermo Fisher Scientific (Villebon-sur-Yvette, France). Immobilon Western Chemiluminescent HRP Substrate was acquired from Merck (Lyon, France). Caspase-3/7 green reagent was purchased from Sartorius (Göttingen, Germany).