Facsaria 2 sorp cell sorter

Prior to antibody staining, cells were incubated for 10 min on ice with TruStain fcX (anti-mouse CD16/32, BioLegend #101319, clone 93, 1:100). To identify infiltrating monocytes/macrophages from the brain, anti-CD11b-PE-Cy7 (BioLegend, M1/70, 1:100), anti-CD45-pacificBlue (BioLegend, 30-F11, 1:100), anti-F4/80-APC (BioLegend, BM8, 1:75) and anti-Ly6C-BV605 (BioLegend, HK1.4, 1:500) were used. Blood-derived monocytes were identified using anti-CD45-pacificBlue (BioLegend, 30-F11), anti-Ly6C-BV605 (BioLegend, HK1.4, 1:100), anti-Cd115-APC (BioLegend AFS98, 1:100) and anti-Cd11c-APC-Cy7 (BioLegend, N418, 1:100). Cells were stained by incubation with antibodies for 30 min on ice. Finally, cells were washed using 1 ml PBS and centrifuged at 400 × g for 8 min. Cell pellets were resuspended in 300 µl PBS supplemented with 0.2% FBS and filtered through a 35 µm cell strainer (BD Falcon). Cell subpopulations were finally sorted into RLT buffer (Qiagen) using a BD FACSAria II SORP Cell Sorter.

Cells were harvested with trypsin and made single cell prior to FACS assays. All sorting experiments using FACS were performed on a BD FACS ARIA™ II SORP Cell Sorter (BD Biosciences). All FACS analysis experiments were performed on a BD FACSCanto™ II (BD Biosciences). Unstained or untransduced wildtype (WT) cells functioned as negative controls. 7-AAD (Cat. #559925, BD Biosciences) at a 1:100 dilution was added to samples to exclude dead cells. FACSDiva v8 software was used to acquire data, data analysis was performed with FlowJo software.
For the Nicoletti assay, 190,000 (HuTu-80) and 125,000 (MDST8) cells were plated per well of a 6 well plate. Medium was collected and combined with cells harvested using trypsin 48 hr. later. Cells were pelleted, taken up in freshly prepared Nicoletti buffer (50 μg/ml propidium iodide, 0.1% sodium citrate and 0.1% Triton X-100 in ddH₂O) and left at 4 °C overnight before analysis on BD FACS Canto II.

Samples from aerobic 100-ml cultures in 500-ml shake flasks were vortexed thoroughly to disrupt cell aggregates and used for flow cytometry on a BD FACSAria II SORP cell sorter (BD Biosciences, Franklin Lakes, NJ) equipped with 355-, 445-, 488-, 561-, and 640-nm lasers and a 70-μm nozzle and operated with filtered FACSFlow (BD Biosciences). Cytometer performance was evaluated prior to each experiment by running a CST cycle with CS&T beads (BD Biosciences). The fluorophore mRuby2 was excited by the 561-nm laser, and emission was detected through a 582-nm bandpass filter with a bandwidth of 15 nm. The fluorophore Venus was excited by the 488-nm laser, and emission was detected through a 545-nm bandpass filter with a bandwidth of 30 nm. For each sample, 10,000 events were analyzed, and the same gating strategy was applied to all samples from the same culture. The reference sample for no fluorescent cells was a mid-exponentially growing culture of IMX585 on SMG. The Venus and mRuby2 fluorescence reference was obtained from mid-exponential aerobic cultures on SMG of IMX2240 or IMX2212 and IMX2238, respectively. Cells without fluorescence and doublets or with Venus and mRuby2 fluorescence were selected in a Venus/mRuby2 plot.

T lymphocytes were mechanically dissociated from thymus using frosted glass slides in DMEM with 5% FBS. Red blood cells were lysed using ACK buffer, and passed through 40 μm mesh filter. T cells were incubated with Fc Block (BD Biosciences) in 3% fetal bovine serum (FBS) (v/v) in PBS for 20 minutes on ice. 1–2 million cells were incubated with or without fluorescent-conjugated antibodies that recognize CD4, CD8, CD45, B220, or isotype control antibodies (BD Biosciences) in the dark for 30 minutes at 4°C. Cells were washed 3x and resuspended in 1% FBS (v/v) in PBS. For FACS sorting, CD45 stained cells were sorted using BD FACSAria II SORP cell sorter (BD Biosciences). CD45+ T cells and CD45- thymic stromal cells were subjected to RNA extraction using Ambion RiboPure RNA purification Kit (Thermo Fisher Scientific) and RT-PCR (Supplementary Methods). For CD4 and/or CD8-stained cells, they were fixed with paraformaldehyde at a final concentration of 1% (v/v). Cells were then run on Dako CyAn ADP flow cytometer or BD FACSCanto II Analyzer, and analyzed using FlowJo software (FlowJo, LLC., Ashland, OR). At least 30,000 viable events were collected for analysis.

FACS of NSCs was performed according to the protocol described by Yuan et al. [29 (link)] at the Human Embryonic Stem Cell Core Facility at Sanford Consortium for Regenerative Medicine (2880 Torrey Pines Scenic Dr., 92037, La Jolla, CA) using BD FACS ARIA II SORP cell sorter (BD Biosciences, Franklin Lakes, NJ, USA). After sorting of CD184⁺/CD271⁻/CD44⁻/CD24⁺ NSCs, cells were plated on P/L-coated cell culture dishes in the density 25,000/cm².
Expression of extracellular and intracellular markers was determined using flow cytometry on fixed cell samples using BD LSRFortessa™ (BD Biosciences, USA). All buffer compositions are listed in Additional file 1: Table S1. All antibodies and corresponding isotype controls are listed in Additional file 2: Table S2).