Cell cycle distribution was analysed by flow cytometry (Becton Dickinson). Cells were synchronized in growth factor-free keratinocyte-SFM with 2 mM thymidine (catalogue no.T1895, Sigma-Aldrich) for 24 h. Transfection is performed using Lipofectamine TM RNAiMAX (Invitrogen) according to its manual, and the final concentration of annealed oligo RNAs is 400 nM. Four hours after transfection, culture medium was replaced by complete keratinocyte-SFM supplemented with L -glutamine, prequalified human recombinant epidermal growth factor (EGF) 1-53 and BPE. Cells were harvested at different time points, washed in PBS, fixed with ice-cold 70% ethanol and strained in PI/RNase solution (BD Pharmingen). The samples were analysed on a FACScan flow cytometer in combination with BD lysis software (Becton Dickinson).
Lysis software
Manufactured by BD
BD lysis software is a computer program designed to facilitate the processing and analysis of biological samples. It provides tools for the lysis, or disruption, of cells or tissues to release their contents for further examination. The software's core function is to enable the efficient and standardized preparation of samples for various downstream analyses, such as protein or nucleic acid extraction.
Automatically generated - may contain errors
Lab products found in correlation
Flow cytometry, by BD (2 mentions)
Facscan flow cytometer, by BD (2 mentions)
Pi rnase solution, by BD (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Thymidine, by Merck Group (1 mentions)
Annexin 5 fitc pi, by BD (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Facscan, by BD (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
4 protocols using lysis software
1
Cell Cycle Analysis by Flow Cytometry
2
Cell Cycle and Apoptosis Analysis
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Cell cycle and apoptosis analyses were performed as described previously.10 (link), 29 (link), 30 (link) In brief, cell cycle distribution and fluorescein isothiocyanate (FITC) Annexin V/PI (BD Pharmingen) for the apoptosis assay were analyzed by flow cytometry (Becton Dickinson), and DNA content and percentage of apoptosis cells were analyzed on a FACScan flow cytometer in combination with BD lysis software (Becton Dickinson).
3
Quantifying Apoptosis and Cell Cycle
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For analysis of apoptosis, ESCC cells were stained by annexin V-FITC and propidium iodide to determine the percentage of apoptotic cells using a kit from BD Biosciences (Bedford, MA, USA). Each sample was then analyzed with flow cytometry (BD FACSCanto II Flow Cytometer; BD Biosciences).
For cell cycle analysis, cells were washed twice with PBS, and fixed in 70% ethanol overnight at 4 °C. The fixed cells were pelleted by centrifugation at 2000×g for 10 min, re-suspended in PBS, stained with 10 mg/mL propidium iodide for 5 min, and incubated in the dark at 4 °C for 30 min. The DNA content was determined using a flow cytometer (Model 100, Beckman Coulter, Kraeme, CA, USA) with excitation and emission settings of 488 and 610 nm, respectively. The proportion of G2/M cells was calculated based on the relative DNA content, as measured by the flow cytometer using LYSIS software (Becton–Dickinson).
For cell cycle analysis, cells were washed twice with PBS, and fixed in 70% ethanol overnight at 4 °C. The fixed cells were pelleted by centrifugation at 2000×g for 10 min, re-suspended in PBS, stained with 10 mg/mL propidium iodide for 5 min, and incubated in the dark at 4 °C for 30 min. The DNA content was determined using a flow cytometer (Model 100, Beckman Coulter, Kraeme, CA, USA) with excitation and emission settings of 488 and 610 nm, respectively. The proportion of G2/M cells was calculated based on the relative DNA content, as measured by the flow cytometer using LYSIS software (Becton–Dickinson).
4
Flow Cytometric Analysis of Apoptosis in Aging Rat Hearts
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Flow cytometric analysis was carried out on FACScan (Becton Dickinson) using fluorescence 1 (FL1), 4 decades (logarithmic), detector 648 V, log amplifier, compensation 1.1%; fluorescence 2 (FL2), 4 decades (logarithmic), detector 496 V, log amplifier, compensation 22.8%. Data analysis was performed using lysis software (Becton Dickinson). Heart specimens were carried out, and cell suspension was prepared with Tris –EDTA buffer (pH 74) (Sigma–Aldrich Co.). They were fixed in ice-cold 96–100 % ethanol (Sigma) at 4 °C overnight, centrifuged at 1500 rpm for 10 min, and suspended in PBS containing 50 μg/mL propidium iodide (PI) (Sigma–Aldrich Co.). Single cell suspensions were prepared from at least of five samples of each of the aging rats, and 1.5–3 × 106 cells were stained with fluorescein isothiocyanate-conjugated annexin V (annexin V-FITC) and assayed after incubation for 15 min at room temperature.
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