Live EGFP NSCs were sorted using an Influx v7 Sorter (BD Biosciences). BD FACS Sortware 1.0.0.650 was used to establish a 4×16 grid over the surface of a dried scWestern slide for deposition of sorted cells. 10 µm polystyrene fluorescent microspheres (Flow-Check Fluorospheres, 6605359, Beckman Coulter) were test-sorted for fine positioning adjustment. Cells were gated for EGFP expression, and sorting was calibrated so that each droplet exiting the nozzle contained a single EGFP-positive cell or no cells. The sorting purity was ~96%. After FACS, gel slides were rehydrated by immersion in PBS and analyzed by scWestern. For propidium iodide (PI) cell staining, PI (1mg/mL, P3566, Life Technologies) was added to cell suspensions at 1:100 dilution. Dead cells were imaged after drying of FACS droplets on scWestern slides by fluorescence microscopy.
Facs sortware 1
Manufactured by BD
BD FACS Sortware 1.0.0.650 is a software application developed by Becton, Dickinson and Company (BD) for flow cytometry data analysis. The software provides users with tools to acquire, analyze, and sort data from BD flow cytometry instruments.
Automatically generated - may contain errors
Lab products found in correlation
Flow check fluorospheres, by Beckman Coulter (3 mentions)
P3566, by Thermo Fisher Scientific (3 mentions)
Influx v7 sorter, by BD (3 mentions)
Mouse fcr blocking reagent, by Miltenyi Biotec (2 mentions)
Lsrfortessa, by BD (2 mentions)
Influx cell sorter, by BD (2 mentions)
Facsjazz, by BD (1 mentions)
Accuri c6 plus, by BD (1 mentions)
6 protocols using facs sortware 1
1
Single-Cell Western Blotting of EGFP-Positive NSCs
2
Single-Cell Western Blotting of EGFP-Positive NSCs
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Live EGFP NSCs were sorted using an Influx v7 Sorter (BD Biosciences). BD FACS Sortware 1.0.0.650 was used to establish a 4×16 grid over the surface of a dried scWestern slide for deposition of sorted cells. 10 µm polystyrene fluorescent microspheres (Flow-Check Fluorospheres, 6605359, Beckman Coulter) were test-sorted for fine positioning adjustment. Cells were gated for EGFP expression, and sorting was calibrated so that each droplet exiting the nozzle contained a single EGFP-positive cell or no cells. The sorting purity was ~96%. After FACS, gel slides were rehydrated by immersion in PBS and analyzed by scWestern. For propidium iodide (PI) cell staining, PI (1mg/mL, P3566, Life Technologies) was added to cell suspensions at 1:100 dilution. Dead cells were imaged after drying of FACS droplets on scWestern slides by fluorescence microscopy.
3
Single-cell Western Blotting of EGFP-Positive Cells
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Live EGFP NSCs were sorted using an Influx v7 Sorter (BD Biosciences). BD FACS Sortware 1.0.0.650 was used to establish a 4×16 grid over the surface of a dried scWestern slide for deposition of sorted cells. 10 µm polystyrene fluorescent microspheres (Flow-Check Fluorospheres, 6605359, Beckman Coulter) were test-sorted for fine positioning adjustment. Cells were gated for EGFP expression, and sorting was calibrated so that each droplet exiting the nozzle contained a single EGFP-positive cell or no cells. The sorting purity was ~96%. After FACS, gel slides were rehydrated by immersion in PBS and analyzed by scWestern. For propidium iodide (PI) cell staining, PI (1mg/mL, P3566, Life Technologies) was added to cell suspensions at 1:100 dilution. Dead cells were imaged after drying of FACS droplets on scWestern slides by fluorescence microscopy.
4
Flow Cytometry Analysis of amyE::PyvmC-yfp
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For flow cytometry analysis of the amyE::PyvmC-yfp reporter, cells grown for 24 h were removed from the agar surface with a P200 tip and placed into an Eppendorf tube containing 1 mL of PBS. Cells were sonicated with an amplitude of 30% between 30 and 60 s without pause until homogeneity, then centrifuged at 13,800 × g for 2 min before adding 200 µL of paraformaldehyde 4%. Cells were fixed for 7 min and washed twice with PBS before analysis. Data collection was performed with BD FACSJazz using a 488-nm laser and BD Accuri C6 plus. Data analysis was achieved with BD software (BD CSampler plus version 1.0.23.1 and BD FACS Sortware 1.2.0.142).
5
Mouse Lung Single-Cell Flow Cytometry
6
Mouse Lung Single-Cell Flow Cytometry
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Mouse lung single-cell suspensions prepared as above were incubated with mouse FcR Blocking Reagent (Miltenyi Biotec) for 10 min at 4°C followed by incubation with fluorescently-conjugated antibodies for 30 min at 4°C (see Supplementary Table 1 for antibody list). Cells were washed twice with MACS buffer, then incubated with 4′,6-diamidino-2-phenylindole (DAPI) to stain dead cells. Flow cytometry analyses were performed on a BD LSR-Fortessa (BD Biosciences) and subsequently analysed using FlowJo 10.4.2 (FlowJO, LCC 2006-2018). Cell sorting was performed on a BD Influx cell sorter (BD Biosciences) using the BD FACS™ Sortware 1.2.0.142.
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