Fcs express 6 flow software

The day before FACS experiments, 0.5x10⁶ RPE-1 cells were seeded onto each well of a 6-well plate containing 2 ml of the RPE-1 growth media per well. On the next day, Hoechst 33342 (EMP Biotech) was added to the media at the final concentration of 2 μM and incubated at 37°C with 5% CO₂ for 60 min before the cells were trypsinized and pelleted. The cells were re-suspended in 500 μl of PBS and analysed by flow cytometry using an iCyt EC800 cell analyser (Sony Biotechnology). The resulting cell-cycle distribution of cell singlets was determined using FCS EXPRESS 6 Flow software (De Novo Software).

Cells were collected by trypsinization and stained against a marker of undifferentiated mESCs, Pecam-1 (BD Biosciences, APC-conjugated, 551262; 1:200), or a marker for PrE differentiated cells, PDGFRA (BD Biosciences, APC-conjugated, 562777; 1:200), and DAPI (Molecular Probes, D1306, 1 μg/ml) to exclude dead cells. mESCs were stained for 15 min at 4°C before being washed and resuspended in FACS buffer (10% Fetal Calf Serum (FCS) in phosphate-buffered saline) with DAPI. Flow cytometry analysis was carried out using a BD LSR Fortessa, and flow cytometry sorting was carried out in a BD FACS Aria III. Data analysis was carried out using FCS Express 6 Flow software (De Novo Software) by gating on forward and side scatter to identify a cell population and eliminate debris, then gating DAPI negative, viable cells before assessing the levels of GFP, mCherry, or APC.

Cells were analysed on a BD Accuri C6 (Becton Dickinson,) and/or a BD FACSCalibur^TM (Becton Dickinson) flow cytometer. Cell debris was discriminated by the forward scatter (FSC) and side scatter (SSC) properties of the cells, and cell aggregates were removed from the analysis by the selection of single cells using the area versus high signal of FSC, as described in Supplementary Fig. 6a. Between 5,000 and 20,000 events were collected in the single scatter gate region and data were analysed with FCS Express 6 Flow software (De Novo Software). The level of positive staining was expressed as the median fluorescence intensity or as a stain index by weighting the fluorescence with the standard deviation of each sample. Negative controls were unlabelled cells. Compensation was performed using antibody capture beads (the compensation matrix can be found in Supplementary Fig. 6b). Gates were placed based on single-labelled controls and by establishing 0.1% as the cutoff point.

Dead cells existing prior to the experiments were removed using Histopaque-1077 (Sigma) or Dead Cell Removal kit (Miltenyl Biotec). Freshly isolated intact iBCBL-1 cell lines were treated with 1 µg/ml Dox for the indicated times. Cell viability was then measured using trypan blue exclusion assays. For apoptosis assays, cells were analyzed by FITC-annexin V staining (BioLegend) or terminal deoxynucleotide transferase (TdT)-mediated dUTP nick labeling (TUNEL) assay kit (AAT Bioquest). All imaging and quantitation of apoptotic cells were performed using Cellometer Vision CBA Image Cytometry (Nexcelom), which employs four independent images for cell counting. FCS Express 6 Flow software (De Novo Software) was used for data analysis.

Retinas were harvested 3 weeks post-injection and trypsin-dissociated, as previously described (Palfi et al., 2012 (link)). To isolate RGCs, cells were labeled with anti-Thyl-PE-Cy5, (CD90.2, Rat Thy1.2, 53-2.1 1:100; eBioscience Inc., San Diego, CA). DRAQ5^TM (BioStatus, Leicestershire, UK) was used to sort nucleus-positive cells after which cell populations were sorted on the basis of forward and sideways scatter, and subsequently two stages of singlet selection. From these, retinal cells expressing both EGFP and Thy1 were identified (BD FACSAria IIIu high speed cell sorter, BD Bioscience, San Jose, CA). EGFP was excited by a 488 nm laser and the emission was collected using a 530/30 band pass filter. Thy-1 PECy5 had been measured exciting the probe with a 561 nm laser and collecting the signal with a 690/40 nm band pass. QC of the cell sorter had been done with BD CS&T beads and the drop delay had been adjusted using the BD Accudrop beads (RUO), following manufacture specifications. EGFP-positive cells expressing Thy1 were represented as a percentage of the total EGFP positive cells. Data has been reanalyzed with the FCSExpress 6 Flow software (DeNovo Software). Statistical analysis (Student's t-test) was performed using Microsoft Excel and p < 0.05 was considered statistically significant.

About 2 × 10⁵ MTHFD2 KD and scRNA rSCC-61 cells were seeded into 6-well cell culture plates, allowed to attach, incubated overnight in serum-free medium, and then irradiated (2 Gy or sham) and treated with β-lap (3 μM) or vehicle control for 2 h. After completion of the incubation period, the culture media was replaced with fresh serum-free media and the cells were incubated for another 22 h. At the end of the incubation period, the cells were washed with PBS, trypsinized, and centrifuged at 200 × g for 5 min. The cells were again washed with PBS, fixed with ice-cold 70% ethanol for 30 min at 4°C, further washed with PBS (two times), and incubated with PBS containing 100 μg/mL RNase A and 50 μg/mL propidium iodide. The cells were washed three times with PBS and resuspended into 300 μL FACS buffer and subjected to flow cytometry using a BD Accuri 6 for analysis. Data analysis to quantify the cell cycle distribution was performed with FCS Express 6 Flow software (De Novo Software, Pasadena, CA, USA).

Cells were fixed with 1% paraformaldehyde and incubated with phycoerythrin (PE)-conjugated anti-human CD105 (Immunostep, 105PE-100T; 1:50) or allophycocyanin (APC)-conjugated anti-human CD166 (Immunostep, 1399990314; 1:50) for 30 min at 4 °C. Events were collected on a FACScalibur^TM flow cytometer (Becton Dickinson) with the CellQuest^TM Pro software. Forward scatter (FSC) and side scatter (SSC) were used to gate alive cells and discriminate cell debris. Data were analysed with FCS Express 6 Flow software (De Novo Software); n = 3 was used for flow cytometry analysis.