For cell cycle analysis, NPCs were seed on matrigel-coated plates at a density of 80,000 cells/cm2. Twenty-four hours after seeding, the cells were detached with Accutase solution, harvested, and fixed in 70% ethanol on ice for 2 h; after washing with PBS, the fixed cell was treated with RNase A (200 μg/ml, Carlo Erba) and stained with propidium iodide (50 μg/ml, ThermoFisher Scientific) at 37 °C in the dark for 20 min. Samples were immediately acquired on FACS Canto II (BD) flow cytometer, and cell cycle profiles were analyzed using FCS Express 6 Flow (De NovoSoftware) and expressed as a percentage. In order to analyze G1/S exit, cells were synchronized by double thymidine block and analyzed by FACS at different time points after thymidine release89 (link). Briefly, 24 h after seeding thymidine (2 mM, Sigma-Aldrich) was added to the culture medium, and cells were cultured for 18 h; then thymidine was removed and cells were released for 9 h in fresh NPC medium. The second round of thymidine (2 mM) was added for 18 h. Then cells were released by washing with 1× PBS and incubating them in fresh NPC medium. Cells were collected at 0, 2, 10, and 24 h after release for propidium iodide staining and FACS analysis as described before.
Fcs express 6 flow
Manufactured by De Novo Software
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FCS Express 6 Flow is a comprehensive software solution for the analysis of flow cytometry data. It provides a user-friendly interface and a wide range of analysis tools to facilitate the interpretation of flow cytometry results.
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Lab products found in correlation
Propidium iodide, by Thermo Fisher Scientific (3 mentions)
Lsrii flow cytometer, by BD (2 mentions)
Human fc block, by BD (2 mentions)
Cyflow cube 6, by Sysmex (2 mentions)
Facscanto 2, by BD (1 mentions)
Facs caliber flow cytometer, by BD (1 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
Myelin removal beads, by Miltenyi Biotec (1 mentions)
Fixation buffer, by BioLegend (1 mentions)
Intracellular staining permeabilization wash buffer, by BioLegend (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Nos2 pe, by Santa Cruz Biotechnology (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Facsverse cytofluorometer, by BD (1 mentions)
10 protocols using fcs express 6 flow
1
Cell cycle analysis of neural progenitor cells
2
Mitochondrial Function Evaluation by Flow Cytometry
3
Flow Cytometry Analysis of Cell Surface Proteins
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Cells were harvested for analysis using TrypLE (Invitrogen) disassociation buffer and collected in 100 μL of 0.1% bovine serum albumin. After collection, cells were incubated with conjugated antibodies against proteins of interest for 30 min on ice. Cells were washed three times with 0.1% BSA and passed through a 40 μm cell strainer. Flow analysis was conducted on a BD FACSCaliber flow cytometer. Following the manufacturer’s instructions, dead cell populations were gated out with forward-side scatterplots. All analyses were conducted using IgG-PE or IgG-FITC (BD) isotype controls. All analyses were performed using FCS Express 6 Flow (De Novo Software, Pasadena, CA). The gating strategy is outlined in Supplementary Fig 6 .
4
Mitochondrial Function Evaluation by Flow Cytometry
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Cells were seeded in 60 mm dishes and cultured in DMEM containing 10% FBS. Cells were washed once with FBS free medium. Mitotracker Red (LifeTecnologies, M7212) was added to a final concentration of 20–40 nM and incubated for 20–30 min at 37°C. Cells were then quickly washed with PBS, trypsinized, collected in phenol red free medium and incubated for 10 min at 37°C in the dark prior to analysis. Unstained cells were negative control. Acquisition was performed on LSR II Flow Cytometer (BD Biosciences, San Jose, CA) on the basis of Forward and Sideward Scatter parameters and texas red fluorescence using BD FACSDiva software. Eight to ten thousand events from each sample were evaluated. Data were analyzed using the FCS Express 6 Flow software (De Novo Software).
5
NG2-EYFP Cell Cycle Analysis
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NG2-EYFP mice were used for cell cycle analysis in the NG2 population, at the ages detailed in the manuscript. For animals older than three weeks, myelin debris were removed from single-cell suspensions with myelin removal beads (#130-096-733, Miltenyi Biotec) according to manufacturer’s instructions. Cells were fixed in Fixation Buffer (BioLegend) for 15 min, permeabilized with Intracellular Staining Permeabilization Wash Buffer (BioLegend) for 10 min. After washing, cells were stained with DAPI (1μg/ml) for 10 min. Cells were washed again and acquired on a BD LSRFortessa flow cytometer (BD Bioscience) equipped with four lasers, a blue (488-nm), a red (640-nm), a violet (405-nm) and a yellow/green (561-nm) laser. The EYFP positive cells were detected by filtration through a 530/30 nm band pass (BP) filter and DAPI through a 450/50 nm BP filter. Analysis was performed with FCS Express 6 Flow (De Novo Software) (Figure S2 ).
6
CFSE-based T-cell Proliferation Assay
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Purified T-cells were subjected to CFSE-based proliferation assay, as previously reported (7 (link), 10 (link)). Briefly, purified T-cells from immunized and re-challenged mice were labeled with CFSE (5 µM) by incubating for 20 min at 37°C in a 5% CO2 humidified atmosphere. CFSE labeled T-cells (0.5×106) were co-cultured with APCs (0.5×106) and stimulated with rMOMP (5 μg/mL) in round-bottom polypropylene tissue culture tubes and incubated for 120 h at 37°C. After incubation, the cells were harvested and stained using CD3-APC-Cy7, CD4-PerCP-Cy5.5, CD62L-APC, and CD44-PE to evaluate T-cell proliferation and memory (CD44high CD62Lhigh) and effector (CD44high CD62Llow) phenotypes. Stained cells were washed, fixed, and data were acquired on a BD LSR II flow cytometer and analyzed using FCS Express 6 FLOW (De Novo Software, Pasadena, CA). Gating on CFSE+ T-cells was used for the selection of CD3+CD4+ T-cell populations. Histogram fluorescence intensities were used to quantify the proliferating and resting T-cells amongst the total CFSE+CD3+CD4+ T-cells.
7
Quantifying c-Kit Expression on LAD2 Cells
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To demonstrate antibody binding to c-Kit on the cell surface of LAD2 cells, flow cytometry assay was performed. LAD2 cells were starved of SCF for 24 h, because SCF can cause internalization and degradation of c-Kit. Cells were rinsed with phosphate-buffered saline (PBS) and blocked with PBS containing 5% bovine serum albumin (BSA) at 4 °C for 1 h. After blocking with BSA, Human BD Fc Block™ (2.5 μg/106 cells, BD Biosciences, CA, USA) was treated to block binding of the antibody to the Fc receptor. The cells (2 × 105 cells) were stained with 2G4, 4C9, or normal human IgG1 (Sino Biological, Beijing, China) at the indicated concentrations at 4 °C for 1 h. The cells were then rinsed thrice in PBS containing 2% BSA and stained with goat anti-human IgG secondary antibody (0.3 μg/mL, Invitrogen, CA, USA) at 4 °C for 1 h. After washing, the fluorescence signal was detected using CyFlow Cube6 (Sysmex Partec, Goerlitz, Germany), and the data analysis was performed using FCS Express 6 Flow (De Novo software, CA, USA).
8
Macrophage Phenotypic Profiling by FACS
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Macrophages were stimulated (section 2.3 above), washed and blocked with Fc blocking Ab (BD Biosciences) in fluorescent-activated cell sorting (FACS) buffer (PBS, 0.1% NaN3, 1.0% FBS for 15 min at 4°C [11 (link), 12 (link)]. The cells were washed and stained with fluorochrome-conjugated antibodies (Abs) (SOCS1-Alexa Fluor® 488, SOCS3-Alexa Fluor® 647, NOS2-PE and MRC1/CD206-Alexa Fluor® 680 (Santa Cruz Biotechnology, Dallas, TX, USA)) for 30 min at 4°C, and then washed, fixed with 2% paraformaldehyde solution (PFA) for 20 min at 4°C. Data were acquired on a BD FACS Canto II flow cytometer (BD Bioscience) with at least 1 × 105 events for each sample and analyzed using FCS Express 6 FLOW (De Novo Software, Pasadena, CA, USA).
9
Flow Cytometry Acquisition and Analysis
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Cytofluorometric acquisitions were performed by means of a FACSVerse cytofluorometer (BD Biosciences). Data were statistically evaluated using the FCS Express 6 Flow (De Novo Software, CA, USA) software.
10
Quantifying c-Kit Receptor Binding
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To demonstrate antibody binding to c-Kit on the cell surface of LAD2 cells, ow cytometry assay was performed. LAD2 cells were starved of SCF for 24 h, because SCF can cause internalization and degradation of c-Kit. Cells were rinsed with phosphate-buffered saline (PBS) and blocked with PBS containing 5% bovine serum albumin (BSA) at 4°C for 1 h. After blocking with BSA, Human BD Fc Block™ (2.5 µg/10 6 cells, BD Biosciences, CA, USA) was treated to block binding of the antibody to the Fc receptor. The cells (2 × 10 5 cells) were stained with 2G4, 4C9, or normal human IgG1 (Sino Biological, Beijing, China) at the indicated concentrations at 4°C for 1 h. The cells were then rinsed thrice in PBS containing 2% BSA and stained with goat anti-human IgG secondary antibody (0.3 µg/mL, Invitrogen, CA, USA) at 4°C for 1 h. After washing, the uorescence signal was detected using CyFlow Cube6 (Sysmex Partec, Goerlitz, Germany), and the data analysis was performed using FCS Express 6 Flow (De Novo software, CA, USA).
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