Cells at a density of 3 × 105 cells/well were seeded into a 6-cm Petri dish and incubated with completed medium at 37 °C for 24 h. Subsequently, the cells were treated with Huaier extract for 48 h, and the total cells were collected. The cells were fixed with 75% cold ethanol (1 mL of PBS and 3 mL of absolute ethanol) at −20 °C overnight. Then, the DNA of cells was stained with 200 of μL RNase A (1 mg/mL) and 500 μL of propidium iodide (PI, 100 μg/mL) (Liankebio, Zhejiang, China) for 30 min at room temperature in the dark, and they were analysed using a FACScan flow cytometer. The data were analysed using ModFitLT software, version 2.0 (Becton Dickinson, Franklin Lakes, NJ, USA).
Propidium iodide
Manufactured by Liankebio
Sourced in China
Propidium iodide is a fluorescent dye that binds to DNA. It is commonly used in flow cytometry and other cell-based assays to stain and quantify cellular DNA content.
Automatically generated - may contain errors
Lab products found in correlation
Facscan flow cytometer, by Liankebio (2 mentions)
Rnase a, by Liankebio (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Facscantoii flow cytometry, by BD (1 mentions)
Facscanto ow cytometer, by BD (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
Pe annexin 5 apoptosis detection kit, by BD (1 mentions)
C myc, by Abcam (1 mentions)
6 protocols using propidium iodide
1
Cell Cycle Analysis of Huaier Extract
2
Cell Cycle Analysis by Flow Cytometry
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3 × 105 cells were seeded into each well on a 6 cm Petri dish the day before. The cells were collected after incubated with Huaier n-butanol extract for 48 h. The cells were fixed with 75% cold ethanol at −20 °C overnight. Then, the cells were stained with 200 of μL RNase A (1 mg/mL) and 500 μL of propidium iodide (PI, 100 μg/mL) (Liankebio, Zhejiang, China) for 30 min at room temperature in the dark, and they were analyzed using a FACScan flow cytometer. The data were analysed using ModFitLT sofware, version 2.0 (Becton Dickinson, Franklin Lakes, NJ, USA).
3
Cell Cycle Analysis of miR-608/RAC2/PAK4 in PCa
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PCa cells transfected with miR‐608 mimic/RAC2 siRNA/PAK4 siRNA were collected 48 hours after transfection and fixed at −20°C overnight in 75% ethanol. Later cells were gathered and treated with propidium iodide (Liankebio). FACSCantoⅡ flow cytometry (BD) and ModFit 4.0 software were used for cell cycle analysis.
4
Annexin V and PI Apoptosis Assay
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Cells were collected after 72 h of transfection or not and washed three times with PBS. Cells were then incubated in dark with FITC-Annexin V and propidium iodide (Liankebio, China) for 15 min. Flow cytometery was performed using FACSCanto ow cytometer (BD, USA) and FlowJo 10.0 software was used to determine the percent of apoptotic cells.
5
Atg22p Localization and Acetic Acid Response
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For Atg22p labeling, cells carrying the plasmid pTEF-ATG22-EGFP were observed using a Zeiss LSM 780 confocal microscope (Carl Zeiss MicroImaging, Göttingen, Germany). The strains were grown and treated with or without acetic acid (150 mM, pH 3.0) for different times as described in the above conditions, then immobilized in the slides prior to confocal microscopy. Propidium iodide (PI) (Lianke, Hangzhou, China) staining was used to analyze the relevance between Atg22p expression and acetic acid-induced cell death. In addition, the positive cells stained by PI and cells with GFP fluorescence were quantified by flow cytometry.
6
Oridonin: Anticancer Compound Characterization
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Oridonin (empirical formula, C20H28O6; molecular weight, 364.43) was purchased from ChemFaces (Wuhan, China). The purity of the oridonin was confirmed by HPLC to be > = 98%. The oridonin was initially dissolved in dimethyl sulfoxide (DMSO) to make a 20 mM stock solution and stored at −20°C for further use. 0.25% trypsin containing EDTA and fetal bovine serum (FBS) was obtained from Gibco (USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Japan). PE Annexin V apoptosis detection kit was from BD Biosciences (San Diego, CA, USA). Cell cycle analysis kit including RNase A and propidium iodide (PI) was purchased from Liankebio (Zhejiang, China). The antibodies against p21, cleaved caspase-3, cleaved PARP, GAPDH, β-actin, LC3B, anti-rabbit/mouse IgG, and HRP-linked antibody were purchased from CST (Danvers, MA, USA). c-Myc and p53 were purchased from Abcam (Cambridge, UK), and AP4 (6B1) was from Abnova (Taipei, Taiwan).
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