Myoblasts were harvested at day 7, washed twice in ice-cold PBS and then fixed by resuspension in ice-cold 70% ethanol and incubated overnight at 4 °C. Cells were then washed in PBS, centrifuged at 300× g and resuspended in PBS. Then RNase and propidium iodide were added to final concentrations of 5 and 50 μg/mL, respectively. After 30-min incubation, propidium iodide fluorescence of 10,000 cells per sample was measured on Guava® easyCyte™ 8HT Benchtop Flow Cytometer (Merck Millipore, Burlington, MA, USA) The obtained data were processed using InCyte 2.7 software (Merck Millipore, Burlington, MA, USA).
Incyte2
Manufactured by Merck Group
Sourced in United States
InCyte2.7 software is a laboratory equipment product developed by Merck Group. It is a data analysis software designed to support the processing and interpretation of experimental data. The core function of InCyte2.7 is to provide users with tools for data management, visualization, and statistical analysis.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Keygen Biotech (3 mentions)
Guava easycyte ht, by Merck Group (2 mentions)
Guava easycyte 8ht benchtop flow cytometer, by Merck Group (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Stempro accutase cell dissociation reagent, by Thermo Fisher Scientific (1 mentions)
Guava flow cytometer, by Merck Group (1 mentions)
Dmem high glucose medium, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Guava easycyte ht flow cytometer, by Merck Group (1 mentions)
Facscan cytometer, by BD (1 mentions)
5 protocols using incyte2
1
Cell Cycle Analysis of Myoblasts
2
Folate Receptor Expression in HeLa and HSC-3 Cells
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The expression of the folate receptor 1 (FOLR1) on the surface of HeLa and HSC-3 cells was examined by flow cytometry. The cells were maintained in DMEM high-glucose medium (ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (ThermoFisher) and penicillin (100 units/ml)/streptomycin (100 μg/ml) (UCSF Cell Culture). To evaluate the effect of folate on the FOLR1 expression, cells were cultured also for 1 week in DMEM without folate (UCSF Cell Culture; Custom Made). The cells were harvested with StemPro® Accutase® Cell Dissociation Reagent (ThermoFisher). After centrifugation for 3 min at 6000 rpm in a microcentrifuge (Eppendorf 5418; USA Scientific, Ocala, FL, USA), the cells were washed twice in PBS containing 2% FBS and 0.05% sodium azide (Sigma) (FACS buffer) and stained at 4°C for 30 min with allophycocyanin (APC)-conjugated anti-FOLR1 or the corresponding APC-conjugated isotype-matched control monoclonal antibody (R&D Systems, Inc., Minneapolis, MN, USA). The cells were post-fixed with 1% paraformaldehyde in FACS buffer and analyzed using a Guava flow cytometer and InCyte 2.7 software (EMD Millipore, Hayward, CA, USA).
3
Cell Cycle Analysis by Flow Cytometry
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To analyze the cell cycle distribution, trypsinized cells were washed with PBS and fixed in 70% ethanol at 4°C overnight. Cells were then washed twice with PBS and incubated with RNase for 30 min at 37°C, followed by incubation with propidium iodide (KeyGEN Biotech) for 30 min at 4°C. The propidium iodide‐stained cells were examined on a Guava EasyCyte HT flow cytometer (Millipore) and analyzed with InCyte2.7 software (Millipore).
4
Cell Cycle and Apoptosis Analysis
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Before analysis of cell cycle distribution, cells were fixed in ice-cold 70% ethanol for 2 hours. After washing with PBS, cells were incubated with RNase (KeyGen Biotech Co. Ltd., Nanjing, China) for 30 min at 37°C followed by incubation with propidium iodide (PI) (KeyGen Biotech) for 30 min at 4°C. The results were examined on a Guava easyCyte HT flow cytometer (Merck-Millipore, Darmstadt, Germany) and analyzed with InCyte 2.7 software (Millipore). For analysis of apoptosis, cells were stained with Annexin V and 7-AAD (both from KeyGen Biotech) according to the manufacturer's instructions. Apoptotic fractions were analyzed using a FACScan cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA). In our studies, the early apoptotic cells (Q4: Annexin V+/7-AAD-staining) and the late apoptotic cells (Q2: Annexin V+/7-AAD+staining) were considered to be undergoing apoptosis, and the numbers of these apoptotic cells as a proportion of total cells were analyzed.
5
Cell Cycle Analysis by Flow Cytometry
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To analyse the cell cycle distribution, trypsinized cells were washed with PBS and fixed in ice-cold 70% ethanol overnight. Cells were then washed 2X with PBS and incubated with RNase for 30 min at 37 °C followed by incubation with propidium iodide (PI) (KeyGEN Biotech) for 30 min at 4 °C. The PI-stained cells were examined on a Guava EasyCyte HT flow cytometer (Millipore) and analysed with InCyte2.7 software (Millipore).
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