Cells were trypsinized and washed with PBS x1, then fixed with 66% ethanol for 2 hours. After fixation, cells were again washed with PBS x1 then stained with propidium iodide (Abcam, Cambridge, UK) by resuspending the cells in 200 μl propidium iodide and RNase staining solution, as per the manufacturer’s protocol. Flow cytometry was performed using an Accuri-C6 flow cytometer. Cell cycle analysis was performed using the ModFit program to determine the percentage of cells in each of the cell cycle phases, for each cell line at each time point (ModFit settings were manual mode, excluding aggregates and debris, with a trapezoid S-phase shape). A FITC pre-conjugated CD44 antibody (CD44-FITC, Miltenyi Biotech, San Diego, CA, USA) was used to stain and quantify CD44+ cells, according to the manufacturer’s instructions. Isotype control antibody Mouse IgG1-FITC (Miltenyi Biotech, San Diego, CA, USA) was used to control for non-specific binding of antibody to cells.
Propidium iodide
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Propidium iodide is a fluorescent dye that binds to DNA and RNA. It is commonly used in flow cytometry and fluorescence microscopy applications to stain and quantify cellular DNA content.
Automatically generated - may contain errors
Lab products found in correlation
Annexin 5 fitc, by Abcam (20 mentions)
Facscalibur flow cytometer, by BD (8 mentions)
Cellquest software, by BD (7 mentions)
Annexin 5 fluorescein isothiocyanate fitc, by Abcam (7 mentions)
Annexin 5 fluorescein isothiocyanate, by Abcam (6 mentions)
Facscalibur, by BD (6 mentions)
Annexin 5, by Abcam (5 mentions)
Facscan flow cytometry, by BD (3 mentions)
Facscalibur system, by BD (3 mentions)
Rnase a, by Merck Group (3 mentions)
Lsrfortessa, by BD (3 mentions)
Annexin 5 fitc apoptosis detection kit, by Abcam (3 mentions)
Facsdiva software, by BD (3 mentions)
Rnase a, by Abcam (3 mentions)
Annexin 5 cy5, by Abcam (2 mentions)
Facscanto 2, by BD (2 mentions)
Cytoflex flow cytometer, by Beckman Coulter (2 mentions)
Flow cytometry, by BD (2 mentions)
Kaluza analysis software, by Beckman Coulter (2 mentions)
Rnase a, by Thermo Fisher Scientific (2 mentions)
115 protocols using propidium iodide
1
Cell Cycle Analysis and CD44 Expression
2
Cytotoxicity Evaluation of Anti-Cancer Compounds
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HepG2 and Huh7 cell lines were obtained from our laboratory for long-term storage. The cells were cultured in DMEM (high glucose) containing 10% fetal bovine serum and 1% penicillin/streptomycin. GSK461364 (S2193), volasertib (S2235), and YM155 (S1130) were purchased from Selleck. A CellTiter-Glo 2.0 cell viability assay kit (G9241) was purchased from Promega. Annexin V-FITC (Cat#: 1001) and propidium iodide (Cat#: 1056) were purchased from BioVision.
3
Optimizing Apoptosis Detection Workflow
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To study cell apoptosis, propidium iodide (BioVision, USA) double staining and Annexin V-fluorescein isothiocyanate staining were performed according to the manufacturer’s instructions. The cell apoptosis was detected by flow cytometric analysis (BD FACSCanto II Flow, Beckman-Coulter, USA). The cells in the upper left area are cells after necrosis, which was reduced to 0% by optimizing operations, such as the speed and time of centrifugation, selection of apoptosis kit, or the door of FSC/SSC.
4
Cell Cycle and Apoptosis Analysis
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For cell cycle analysis, single cell suspensions were fixed with 70% ethanol for 30 min at 4°C followed by RNA digestion with RNAase (0.5 mg/ml). Then, the permeabilized cells were labeled with propidium iodide (5 mg/ml; Sigma-Aldrich, MO, USA). Subsequently, DNA content was assessed by using an Epics xL flow cytometer (Beckman Coulter, U. K.). For the cell apoptosis assay, the cells were stained with APC conjugated anti-AnnexinV antibody and propidium iodide (PI) according to the manufacturer's protocol manufacturer (BioVision Inc., Milpitas, CA, USA). The percentage of AnnexinV+ PI+ cells were determined by using an Epics xL flow cytometer (Beckman Coulter, UK).
5
Apoptosis Evaluation by Flow Cytometry
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A total of 5 × 105 cells were recovered by centrifugation for apoptosis evaluation via double staining with annexin V–fluorescein isothiocyanate (annexin V–FITC) and propidium iodide20 (link) (BioVision, St Lake Worth, FL, USA) according to the manufacturer's instructions, followed by flow cytometric analysis. Cell Quest software (Becton, Franklin Lakes, NJ, USA) was used to determine the percentage of apoptotic cells.
6
Inhibitor-Mediated Cell Growth and Apoptosis
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Cells were incubated with different concentrations of inhibitors for up to 4 days. Stock solutions of inhibitors were made in DMSO, and the final concentration of DMSO in the cell incubation system was controlled at 0.1%. For cell growth analysis, viable cells were enumerated using Trypan blue staining with a cytometer. For cell viability assay, 0.08 mg/ml XTT (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) and 8 μM Phenazine Methyl Sulfate (PMS) were added to the cells, and absorbance at 450 nm was measured after 3 hr incubation at 37°C. For apoptosis analysis, cells were stained with Cy5-annexin V and propidium iodide (Biovision, CA, USA). To assess cell cycle arrest, cells were fixed with ethanol overnight at 4°C and then stained with propidium iodide in the presence of RNase. Flow cytometric analyses were performed by using a FACSCalibur flow cytometer (BD Biosciences, CA, USA) at the Flow and Image Cytometry Laboratory of University of Oklahoma Health Sciences Center.
7
Apoptosis Assessment in HeLa Cells
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HeLa cells, untreated or treated with cobalt chloride and/or hypoxia, were washed with PBS and collected by centrifugation after trypsinization. The cell pellet was suspended in binding buffer (Annexin V-FITC kit; BioVision, Milpitas, CA, United States), followed by the addition of 5 μl Annexin V-labeling solution (BioVision) and 5 μl propidium iodide (BioVision). Cells were then incubated for 5 min at room temperature and analyzed via fluorescence-activated cell sorting (FACS; BD FACSCanto II).
8
Inhibitor-Mediated Cell Growth and Apoptosis
9
Annexin V and PI Staining
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Cells were plated in a 6 cm culture dish at a seeding density of 5 × 106 cells/dish. Following statin treatment, the medium was removed and washed twice with PBS. The cells were trypsinized, collected by centrifugation, resuspended in 500 μL of 1X Annexin V Binding Buffer mixed with 1 μL of Annexin V (BioVision) and 1 μL of Propidium Iodide (BioVision), and incubated at room temperature for 15 min in darkness. The reaction volume was adjusted with the binding buffer to 1 mL. The cells were analyzed using a flow cytometer (Cytomics FC 500) with a single laser emitting excitation light at 488 nm.
10
Annexin V-FITC Apoptosis Assay
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Detached cells
were suspended in Annexin V binding buffer (cat. no. 1006, BioVision
Inc.) followed by staining with the Annexin V-FITC reagent (cat. no.
1001, Biovision Inc.) and propidium iodide (cat. no. P1304MP, Thermo
Fisher Scientific) by incubating cells on ice for 15 min. Analyses
were performed on a Beckman Coulter CytoFLEX flow cytometer. CytExpert
software was used to analyze the data. For statistical analysis, two-way
analysis of variance (ANOVA) followed by Tukey’s multiple comparison
test was used for comparing treatment efficiency between cell lines,
while effects of the treatment within the cell lines compared to corresponding
controls were compared using two-way ANOVA followed by Sidak’s
multiple comparison test. p < 0.05 was considered
as statistically significant.
were suspended in Annexin V binding buffer (cat. no. 1006, BioVision
Inc.) followed by staining with the Annexin V-FITC reagent (cat. no.
1001, Biovision Inc.) and propidium iodide (cat. no. P1304MP, Thermo
Fisher Scientific) by incubating cells on ice for 15 min. Analyses
were performed on a Beckman Coulter CytoFLEX flow cytometer. CytExpert
software was used to analyze the data. For statistical analysis, two-way
analysis of variance (ANOVA) followed by Tukey’s multiple comparison
test was used for comparing treatment efficiency between cell lines,
while effects of the treatment within the cell lines compared to corresponding
controls were compared using two-way ANOVA followed by Sidak’s
multiple comparison test. p < 0.05 was considered
as statistically significant.
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