Cells were grown to ~ 50% confluence prior to treatment. Floating cells were collected from the media and adhered cells were collected using Accutase (Innovative Cell Technologies, San Diego, CA). For cell death analysis, cells were washed with cold PBS and resuspended in FACS binding buffer (PBS, 1% bovine serum albumin fraction V, 25 mM HEPES, 1 mM EDTA) followed by staining with propidium iodide (BD Pharmingen, San Jose, CA). Annexin V was not used since the cells were strongly adherent. For cell cycle analysis, cells were resuspended in cold 0.5% glucose/PBS and fixed in 70% ethanol. For staining with propidium iodide, cells were resuspended in 0.1% triton X-100/PBS along with RNase A (Sigma-Aldrich). FACS analysis was performed on Accuri C6 flow cytometer (Accuri, Ann Arbor, MI) using 100,000 events. Unstained cells were used as controls for setting the population parameters and overlay of histograms shows no deviation or drift of channels. More than a 5% change from the control was considered statistically significant. For cell cycle statistics, data was analyzed using MultiCycle AV (Phoenix Flow Systems, San Diego, CA) with FCS Express plug-in (De Novo, Los Angeles, CA).
Multicycle av
Manufactured by Phoenix Pharmaceuticals
Sourced in United States
The MultiCycle AV is a versatile laboratory equipment designed for automated cell culture and virus amplification. It provides a controlled environment for the cultivation and expansion of cells, as well as the propagation of viruses. The device features programmable temperature, humidity, and gas control to support various cell and virus culture requirements.
Automatically generated - may contain errors
Lab products found in correlation
Click it plus edu alexa fluor 647 flow cytometry assay kit, by Thermo Fisher Scientific (2 mentions)
Accutase, by Innovative Cell Technologies (1 mentions)
Rnase a, by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Propidium iodide, by BD (1 mentions)
Propidium iodide, by Fujifilm (1 mentions)
Cytomics fc500 flow cytometry analyzer, by Beckman Coulter (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (1 mentions)
Propidium iodide, by Dojindo Laboratories (1 mentions)
Epics xl, by Beckman Coulter (1 mentions)
Facscalibur, by BD (1 mentions)
Lsrii analytical flow cytometer, by BD (1 mentions)
8 protocols using multicycle av
1
Quantifying Cell Cycle and Death
2
Cell Cycle Analysis of MCF-7 Cells
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After 24 h of cell seeding, the cells were synchronized with serum free medium for another 24 h. The following day, additives mixed in fresh 10% FCS-containing medium were applied. MCF-7 samples were harvested after three days of 0 or 25 μg/ml IAA treatments. On the third day, the samples were fixed in 70% ethanol at 4 °C for 24 h. The fixed samples were washed with PBS and incubated in 100 μg/ml propidium iodide (Wako, Yokohama, Japan) and 200 μg/ml RNase (Sigma) for 30 min at 37 °C in the dark. The flow cytometric cell analysis was performed, using a Beckman Coulter Cytomics FC500 Flow Cytometry Analyzer (Beckman Coulter, Fullerton, CA, USA). MultiCycle AV (Phoenix Flow Systems) DNA analysis software enabled determination of the phase of cell cycle arrest by comparing percentages of each cell stage between the control and treatment groups (G1, S, G2/M).
3
Annexin V-FITC Apoptosis Assay
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MH7A cells were stimulated with 20 ng/ml TNF-α (PeproTech, Princeton, USA) for 4 h and then treated with different doses of CGA for 24 h. Subsequently, cells were trypsinized and collected for the detection of apoptotic cells using an Annexin V-FITC Apoptosis Detection kit (Cat# 556547, BD Biosciences, USA). Briefly, MH7A cells were washed twice with cold PBS at 4°C and resuspended in 100 μl binding buffer. After stained with FITC-conjugated Annexin V and propidium iodide (PI), cells were incubated for 15 min at room temperature and then analyzed by flow cytometry MultiCycle AV Phoenix Flow Systems (San Diego, CA, USA). All experiments were performed three times.
4
Cell Cycle Profiling and Differentiation Analysis
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Cell cycle profiles were determined by analyzing the DNA contents using propidium iodide (Dojindo, Kumamoto, Japan) staining and flow cytometry. To assess the differentiation, cells were incubated with 1 µM ATRA for various times. Then, the cells were washed with PBS, fixed with ice-cold 70% ethanol, and stored at −20°C until analysis. The fixed cells were collected, suspended in 300 µl of RNase/PBS solution (100 µg/ml) and incubated at 37°C for 30 min. The cells were collected, resuspended in 300 µl of propidium iodide/PBS solution (5 µg/ml), and incubated in the dark at room temperature for 15 min. After filtration through a nylon mesh sheet, all samples were applied to an Epics XL (Beckman Coulter, Nyon, Switzerland). To analyze the data, WinMDI2.9 and MultiCycle AV for Windows (Phoenix, San Diego, CA) were employed as described [15] (link).
5
Cell Cycle and Apoptosis Analysis
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To analyse the cell cycle, the cells were collected and fixed in ice-cold 70% ethanol in PBS and stored at −4°C until use. After resuspension, the cells were incubated with 100 µl of RNase I (1 g/ml) and 100 µl of propidium iodide (400 µg/ml) at 37°C and were then analysed by flow cytometry (FCM; BD Biosciences, San Jose, CA, USA). The cell cycle phase distribution was calculated from the resultant DNA histogram using MultiCycle AV software (Phoenix Flow Systems, San Diego, CA, USA). Apoptosis was detected with an Annexin V-FITC kit according to the manufacturer's instructions (Trevigen, Inc., Gaithersburg, MD, USA). Briefly, the cells that received various treatments were collected and stained with Annexin V-FITC and PI in the dark for 15 min at room temperature. After the addition of binding buffer, the cells were subjected to flow cytometry and analysed using WinMDI 2.9.
6
Suramin-induced Apoptosis Evaluation by EdU Assay
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Apoptosis assay was performed by EdU staining using a Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Life Technologies), according to the manufacturer’s protocol. KATO-III cell line was synchronized with IMDM medium having 5% FBS for 24 hrs. The following day, cells were incubated for 4 days with or without 200 µM suramin and then trypsinized. These harvested cells were incubated in culture medium with 15 µM EdU for 2 hrs. After incubation, cells were washed with 1% BSA in PBS and 100 µl of Click-iT fixative was added for 15 minutes at room temperature. After washing, cells were incubated in Click-iT Plus reaction cocktail including fluorescent dye (Alexa Fluor 647 picolyl azide) for 30 minutes. The flow cytometric cell analysis was performed, using a BD LSR II analytical flow cytometer (Becton Dickinson, San Jose, CA). MultiCycle AV (Phoenix Flow Systems) DNA analysis software enabled determination of the phase of cell cycle arrest by comparing percentages of each cell stage between the control and treatment groups (G1, S, G2/M).
7
Cell Cycle Analysis of A549 Cells
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The cell cycle phases of A549 cells were studied using flow cytometry (FACScalibur, BD Bioscience, Franklin Lakes, NJ) (Alfaifi et al., 2020 ). Six-well plates were utilized to seed the cells in a density of 3 × 105 cells/well. These were divided into two: untreated (control) or treated group (treated with plain (INVA-APA), 2ME, or 2ME-INVA-APA) left for 24 hours. Next, the cells were washed; 70% cold ethanol was used to fix the cells, and RNase staining buffer and propidium iodide (PI) were used to stain them. Flow cytometry resulted in 10,000 gated events. MultiCycle AV was used to analyze the data (Phoenix Flow Systems, San Diego, CA).
8
Cell Cycle Analysis Using EdU Assay
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Cell cycle distribution was analyzed using the Click-iT™ Plus EdUAlexa Fluor 647™ Flow Cytometry Assay Kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The KATO-III cell line was incubated with IMDM and 5% FBS for 24 h. The following day, the cells were treated with or without 4.5 mM acetyl salicylic acid. Following 4 days of treatment, the cells were trypsinized, harvested and incubated in culture medium with 15 µ MEdU for 2 h. After incubation, the cells were washed with 1% BSA in PBS and 100 µl of Click-iT fixative was added for 15 min at room temperature. Following washing, the cells were incubated with the Click-iT Plus reaction cocktail including fluorescent dye (Alexa Fluor 647 picolylazide) for 30 min. The flow cytometric analysis was performed using a BD LSR II analytical flow cytometer (BD Biosciences). MultiCycle AV (Phoenix Flow Systems) DNA analysis software enabled the determination of the phase of cell cycle arrest by comparing the percentages of each cell stage (G1, S and G2/M) between the control and treatment groups.
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