The cells in 0.5 mL of PBS were mixed with 4.5 mL of ethanol 70% and maintained overnight at 4 °C. Cell suspensions were then centrifuged for 10 min at 310× g and resuspended in 0.5 mL of RNAsa solution containing 0.1% Triton X-100, 20 µg/mL of IP and 0.2 mg/mL of RNAsa (Sigma-Aldrich, St. Louis, MO, USA). After 30 min of incubation at 37 °C, PI fluorescence was detected in a FACScan Becton Dickinson flow cytometer with a 585/42 filter, exciting the sample at 488 nm. The CellQuest Program of Becton Dickinson was used to calculate the percentage of cells in each cycle phase: G0/G1 (growth), S (DNA synthesis) and G2/M (growth and mitosis).
Rnasa
Manufactured by Merck Group
Sourced in United States
RNAse is a lab equipment product used for the degradation of RNA molecules. It functions by catalyzing the hydrolysis of the phosphodiester bonds in RNA, breaking down the RNA into smaller fragments.
Automatically generated - may contain errors
7 protocols using rnasa
1
Cell Cycle Analysis by Flow Cytometry
2
Cell Cycle Analysis by Flow Cytometry
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Cells in 0.5 mL of PBS were mixed with 4.5 mL of ethanol 70% and maintained overnight at 4 °C. Cell suspensions were then centrifuged for 10 min at 310× g and resuspended in 0.5 mL of RNAsa solution containing 0.1% Triton X-100, 20 µg/mL of IP and 0.2 mg/mL of RNAsa (Sigma-Aldrich, St. Louis, MO, USA). After 30 min of incubation at 37 °C, PI fluorescence was detected in a FACScan Becton Dickinson flow cytometer with a 585/42 filter, exciting the sample at 488 nm. The CellQuest Program of Becton Dickinson was used to calculate the percentage of cells in each cycle phase: G0/G1 (growth), S (DNA synthesis) and G2/M (growth and mitosis). To quantify the cell apoptosis, the SubG1 fraction (cells with fragmented DNA) was evaluated. A total of 104 cells were analyzed in each sample in order to ensure a correct statistical significance.
3
Cell Cycle Analysis by Flow Cytometry
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Cell suspensions were washed and incubated with 0.5 mL of PBS and 4.5 mL of ethanol 70% for 4 h at 4 °C. After 10 min and 310 g centrifugation, the cells were resuspended in 0.1% Triton X-100, 20 µg/mL propidium iodide (PI, Sigma-Aldrich, St. Louis, MO, USA), and 0.2 mg/mL of RNAsa (Sigma-Aldrich, St. Louis, MO, USA) and incubated at 37 °C for 30 min. PI fluorescence was detected by exciting the sample at 488 nm using a FACScalibur Becton Dickinson flow cytometer (Becton Dickinson, San Jose, CA, USA) with a 585/42 filter. The percentage of cells in each cell cycle phase was calculated using the CellQuest program (Becton Dickinson), using positive and negative controls and studying at least 104 cells in each sample: SubG1, G0/G1, S, and G2/M (fractions indicative of apoptosis, growth, DNA synthesis, and growth/mitosis, respectively).
4
Cell Cycle Analysis of Riluzole-Treated Cells
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Cell cycle analysis was performed as we described elsewhere40 (link). In brief, cells (5 × 105) were seeded in cell culture dishes. After 24 h, the medium was removed and replaced with fresh medium containing riluzole 0–20 µM for 48 h. Cell cycle analysis was performed by measuring the amount of propidium iodide (PI) in ethanol fixed cells. In brief, cells were harvested by trypsinization and fixed with cold 70% ethanol for 24 h. Cells were rinsed 3 times with ice-cold PBS and resuspended in 1 ml of permeabilizing solution (Triton X 100 (0.25%), sodium azide (0.01%) and RNAs A (100 μg/μl Sigma-Aldrich) in PBS for 10 min. Cells were rinsed once with PBS, resuspended with 1 ml of PBS with PI (2.5 mg/ml) and incubated for 15 min at 4 °C. Cell cycle analysis was performed using a flow cytometer (Becton Dickinson).
5
Cell Cycle Analysis by Flow Cytometry
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The cells were trypsinised, washed twice with ice-cold PBS, and finally resuspended in 330 μL of ice-cold PBS, followed by the addiction of 660 μL of ice-cold methanol while vortexing. To measure the samples, these were washed twice with PBS and then resuspended in 0.5 mL of propidium iodide staining solution (500 μL PBS; 5 μL RNAsa (Sigma) 10 uM; 10 μL IP (Sigma) 1 uM). The samples were kept in the dark until they were analysed in the FACSCalibur flow cytometer, using the FL2 channel. The results were analysed with the Cell Quest program.
6
Nanomaterial Cytotoxicity Evaluation in RAW-264.7 Cells
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Cells were resuspended in PBS (0.5 ml) and incubated with 4.5 ml of ethanol 70% during 4 hours at 4 ºC. Then, cells were centrifuged at 310 g for 10 min, washed with PBS and resuspended in 0.5 ml of PBS with 0.1 % Triton X-100, 20 µg/ml of propidium iodide (IP) and 0.2 mg/ml of RNAsa (Sigma-Aldrich, St. Louis, MO, USA). well culture plates (Corning, USA), at a density of 10 5 cells/ml, in 2 ml of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gibco, BRL), 1 mM L-glutamine (BioWhittaker Europe, Belgium), penicillin (200 μg/ml, BioWhittaker Europe, Belgium), and streptomycin (200 μg/ml, BioWhittaker Europe, Belgium) at 37 ºC under a CO 2 (5%) atmosphere. After 24 hours of culture in the presence or the absence of 50 μg/ml of NanoMBGs, the attached RAW-264.7 cells were washed with phosphate buffered saline (PBS), harvested using cell scrapers and counted with a Neubauer hemocytometer for the analysis of cell proliferation. Then, cells were centrifuged at 310 g for 10 min, resuspended in fresh medium for the analysis of viability, cell size and complexity by flow cytometry as described below.
7
Cell Cycle Analysis by Flow Cytometry
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Cell suspensions were centrifuged at 310xg for 10 min, resuspended in PBS (0.5 ml) and incubated with 4.5 ml of ethanol 70% during 4 hours at 4ºC. Then, cells were centrifuged at 310xg for 10 min, washed with PBS and resuspended in 0.5 ml of PBS with Tritón X-100 0,1%, IP 20 mg/ml and RNAsa 0,2 mg/ml (Sigma-Aldrich, St. Louis, MO, USA).
After incubation at 37ºC for 30 min, the fluorescence of PI was excited by a 15 mW laser tunning to 488 nm and the emitted fluorescence was measured with a 585/42 band pass filter in a FACScalibur Becton Dickinson flow cytometer. The cell percentage in each cycle phase: G0/G1, S and G2/M was calculated with the CellQuest Program of Becton Dickinson and the SubG1 fraction was used as indicative of apoptosis.
After incubation at 37ºC for 30 min, the fluorescence of PI was excited by a 15 mW laser tunning to 488 nm and the emitted fluorescence was measured with a 585/42 band pass filter in a FACScalibur Becton Dickinson flow cytometer. The cell percentage in each cycle phase: G0/G1, S and G2/M was calculated with the CellQuest Program of Becton Dickinson and the SubG1 fraction was used as indicative of apoptosis.
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