The effect of NAP9 and NAPSC on cell viability was determined by flow cytometry (FACS scanner, Becton Dickinson) using propidium iodide (RD systems) and annexin V conjugates (Life Technologies), as provided by the manufacturers.
Facs scanner
Manufactured by BD
Sourced in United States
The FACS (Fluorescence-Activated Cell Sorting) scanner is a laboratory instrument used for the analysis and sorting of cells or particles within a fluid sample. It utilizes laser technology and detectors to identify and differentiate cells based on their size, granularity, and fluorescent properties.
Automatically generated - may contain errors
Lab products found in correlation
Cellquest software, by BD (4 mentions)
Propidium iodide, by Merck Group (2 mentions)
Alexa fluor 488 annexin 5 dead cell apoptosis kit, by Thermo Fisher Scientific (2 mentions)
Annexin 5 conjugate, by Thermo Fisher Scientific (1 mentions)
Annexin 5 pi double staining, by Keygen Biotech (1 mentions)
Annexin 5 apoptosis detection kit 1, by BD (1 mentions)
Tcs spe, by Leica (1 mentions)
Cellquest, by BD (1 mentions)
10 protocols using facs scanner
1
Evaluating Cell Viability via Flow Cytometry
2
Apoptosis Analysis by Flow Cytometry
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At 48 hours after transfection, cells were collected and subjected to annexin V/PI double staining according to the manufacturer’s instructions (Keygen Biotech, China). The samples were then analyzed using a FACS scanner (Becton Dickinson) and the FlowJo software (Tree Star, Ashland, OR, USA).
3
Cell Cycle and Apoptosis Analysis
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SGC-996 cells were plated in 6-well plates at a density of 3×105 cells per well and incubated for 24 h at 37°C. For cell cycle analysis, sub-confluent cells were treated with or without various concentrations of TSA (0.1 and 0.4 μM) or SAHA (1, 2.5 and 5 μM) for 48 h. The cells were then harvested, washed twice with ice-cold phosphate-buffered saline (PBS) for cell cycle and apoptosis detection. For cell cycle distribution analysis, cells were fixed with 70% ethanol, treated with 1% RNase, and stained with propidium iodide (100 μg/ml final concentration; Sigma-Aldrich). Cell apoptosis was assayed using Annexin V Apoptosis Detection Kit I (BD Biosciences) according to the manufacturer’s instructions. Cells at a density of 1×106 cell/ml were resuspended with 1× binding buffer and then 100 μl of the resulting cell suspension was mixed with 5 μl Annexin V and 5 μl 7-AAD. The samples were then analyzed for the proportion of apoptotic cells using a FACS scanner (Becton Dickinson) and the software FlowJo (Tree Star, Ashland, OR, USA). Sub-G1 cells identified in flow cytometric histograms were considered apoptotic cells.
4
Osteoblast Apoptosis Quantification
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Osteoblast apoptosis was determined using FITC-labeled Annexin-V. Cell necrosis was determined using 1 μg/mL propidium iodide (PI). After exposure to the various experimental conditions, the cells were trypsinized and labeled with the fluorochromes at 37°C. Cytofluorometric analysis was performed using an FACS scanner (Becton Dickinson, NY, USA).
TUNEL assay was performed to identify the rate of cell apoptosis. For performing the TUNEL assay, the cells were inoculated on a coverslip, fixed with 4% paraformaldehyde in PBS, and permeabilized with 0.2% Triton X-100 in citrate buffer. Next, the cells were incubated with TUNEL reaction mixture at 37°C for 1 h and were counterstained with DAPI. Apoptotic cells were observed by a technician who was blinded to the treatments by using a fluorescence microscope (Leica TCS SPE, Germany). The percentage of apoptotic cells was estimated by counting a total of 300 cells from random fields. We followed the methods of Mao et al. [8 (link)].
TUNEL assay was performed to identify the rate of cell apoptosis. For performing the TUNEL assay, the cells were inoculated on a coverslip, fixed with 4% paraformaldehyde in PBS, and permeabilized with 0.2% Triton X-100 in citrate buffer. Next, the cells were incubated with TUNEL reaction mixture at 37°C for 1 h and were counterstained with DAPI. Apoptotic cells were observed by a technician who was blinded to the treatments by using a fluorescence microscope (Leica TCS SPE, Germany). The percentage of apoptotic cells was estimated by counting a total of 300 cells from random fields. We followed the methods of Mao et al. [8 (link)].
5
Podocyte Nitric Oxide Quantification
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Nitric oxide production was measured in podocytes loaded with diaminofluorescein diacetate (DAF-DA; 2 µM), and propidium iodide (1 µg/sample) was used to determine cell viability as previously described (58). After exposure to different experimental conditions; PBS (control) and BPA (100 nM) for 24 h, cells were and labeled with the fluorochrome at 37 °C for 1 h 30 min, trypsin dispersed and followed by cytofluorometric analysis with a fluorescence-activated cell sorter (FACS) scanner (Becton Dickinson, New York, NY, USA). Ten thousand events were analyzed for each condition.
6
Cell Cycle Analysis by Flow Cytometry
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Cells were seeded in 6-well tissue culture dishes at 1 × 105 cells per well. The cells were allowed to adhere for 24 h and were then incubated with the indicated drug concentrations. Each condition was tested in triplicate wells. The cell cycle phases of the treated cells were determined by quantifying their DNA content through staining with propidium iodide staining, and the cell cycle phases were analyzed by a FACS scanner (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA) and quantified by Cell Quest software (Becton Dickinson Immunocytometry Systems).
7
Cell Cycle and Apoptosis Analysis
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The cell cycles including subG1 (apoptotic phase), G1, S and G2/M of the assayed cells were determined by staining their DNA contents with propidium iodide (Sigma-Aldrich, St. Louis, MN, USA) and quantifying the degree of staining with a FACS scanner and Cell Quest software (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA). At least 10 000 cells were used for each analysis, and results were displayed as histograms.
8
Granulosa Cell Cycle Analysis
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A FACS scanner and Cell Quest software (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA) were applied to determine the cell cycles of the treated cells by quantifying their DNA contents with propidium iodide staining and to determine the purity of granulosa cell by quantifying the FSHR-positive cells with isotype control by using fluorescence intensity detection.
9
Quantifying Cardiomyocyte Apoptosis with Annexin V
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The status of apoptosis of cardiomyocytes was determined by quantifying Annexin V/PI staining. With the FACS scanner and Cell Quest software (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA) according to manufacturer’s instructions from the Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA).
10
Cell Cycle and Apoptosis Analysis
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A FACS scanner and Cell Quest software (Becton Dickinson Immunocytometry Systems, San Jose, CA) were used to determine cell cycle and the status of apoptosis by quantifying Annexin V/PI staining. Staining was quantified according to manufacturer’s instructions from the Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit (Thermo Fisher Scientific Inc.).
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