Live dead near ir

Tumor cells were either pretreated with IFN-γ (100 IU/mL, Peprotech) or left untreated for 3 days. TILs were then added in a 1:1 ratio, with protein transport inhibitors brefeldin A (1:1000 dilution, GolgiPlug, catalog 555029, BD), Monensin (1:1000 dilution, GolgiStop, catalog 554724, BD), and anti–CD107a-BV421 antibodies (clone H4A3, BD 562623). Tumor cells and TILs were cocultured for 5 hours, after which all cells were stained with Near-IR LIVE/DEAD (Life Technologies) and for surface markers CD3-FITC (clone SK7, BD 345764), CD8-QDot605 (clone 3B5, Thermo Fisher Q10009), and CD4-BV711 (clone SK3, BD, catalog 563028). Subsequently, the cells were fixed and permeabilized (eBioscience) overnight and stained for intracellular cytokines TNF-APC (clone MAb11, BD catalog, 554514) and IFN-γ–PE-Cy7 (clone B27, BD, catalog 557643). Cells were analyzed on a Novocyte Quanteon (ACEA Biosciences). See details related to antibodies used in Supplemental Table 2.

Primary cultures of OPCs derived from neonatal rats were expanded and reseeded 500,000cells/well in a 48-well plate. Settled cells were cultured with 10 µM EdU for 72h in media containing Neurobrew, N2 and PDGF-BB as described above. In addition, media was supplemented with full (1/1), half (1/2) of absent (0) of bFGF concentration referred to the concentration described above. Cells were harvested with Accutase, labeled with anti-PDGFRα (Milteny Biotec, Bergisch, Germany). Dead cells were excluded using near IR Live/Dead (Thermo Fisher Scientific, Waltham, MA). Samples were analyzed with a 3-laser Beckman Coulter Gallios using Kaluza Software (Beckman Coulter, Brea, CA).

The ADCC and ADCP assays are also modifications of the CBA in combination with previously established flow cytometry-based ADCC (48 (link)) and ADCP assays (87 (link), 88 (link)). In this case, serum (1:10) or mAbs (1μg/mL unless otherwise indicated) were added to 5,000 MOG-GFP transfected HEK cells plated in 96-well round-bottom plates and incubated for 15 minutes at room temperature. All serum samples were HI prior to use to inactivate endogenous complement proteins. Then, effector cells were added at a 10:1 effector/target ratio. For the ADCC assay, the effector cells were NK cells that were magnetically isolated (EasySep Human NK Cell Isolation Kit, STEMCELL Technologies) from pooled HD-derived cryopreserved PBMCs. The effector cells for the ADCP assay consisted of the THP-1 macrophage cell line (ATCC), labeled with CellTrace Violet (Thermo Fisher Scientific). After addition of the effector cells, the plates were then incubated at 37°C for 4 hours and shaken intermittently. For the ADCC assay, the cells were then stained with Near IR Live/Dead (Invitrogen) stain for 30 minutes on ice, to identify killed target cells. MOG⁺ and MOG^– were detected as in the CBA as GFP⁺. For the ADCP assay, macrophages were identified in the V450 channel and phagocytosis of MOG⁺ cells in the GFP channel. Data were normalized to the mean media-only condition (no antibodies or serum).

After each treatment, cells were washed with ice-cold FACS buffer (PBS with 0.5% FBS) and then stained for 15 min at 4 °C in FACS buffer containing antibodies against human CD34 (BD Biosciences), CD38(BD Biosciences), Lineage cocktail (BD Biosciences), CD123(BD Biosciences), CD90, (BD Biosciences), CD45(BD Biosciences), and Near IR live dead (Invitrogen). The measurements of intracellular proteins were assayed per the Fix Perm kit (BD Biosciences) and stained for RPL5 (ImageBio). For measurement of ROS, CellRox (Invitrogen) was used to measure per manufacturer's protocol. Stained cells were analyzed immediately on a FACS Celesta cytometer (BD Biosciences). In order to isolate specific subpopulations, the cells were stained as above and a FACS Aria II (BD Biosciences) was used to viably sort cells for further assays. Data were analyzed and prepared via FlowJo^TM (Treestar).