Cells (1×106 cells/well) were seeded on 6 well plates (Corning Life Sciences, Shanghai, China) and cultured with DMEM medium at 37°C with 5% CO2 for 24 h, prior to being transfected with 100 nM HSP70-siRNA or control siRNA (siCON) for 48 h. For the flow cytometry, cells were trypsinized, pelleted via centrifugation at 1,000 × g for 5 min at 4°C and resuspended in 300 µl 0.1% Triton X-100 (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany)/PBS. Cells were fixed with the cold 70% ethanol (Sangon Biotech Co., Ltd., Shanghai, China) at 4°C for 2 h and centrifuged at 1,000 × g for 5 min at 4°C. Subsequent to washing twice in cold PBS and centrifugation at 1,000 × g for 5 min at 4°C, cells were treated with RNase Type I-A (Sigma-Aldrich; Merck KGaA) at 37°C for 15 min and stained with PI (1 mg/ml) at room temperature for 10 min in the dark. Cellular DNA content was determined using a FACSCalibur™ (BD Biosciences). Cell cycle phase distributions were analyzed by ModFit LT™ (v3.0; BD Biosciences) cell cycle analysis software.
Rnase type 1 a
Manufactured by Merck Group
Sourced in Germany, United States
RNAse (Type I-A) is a laboratory enzyme used to degrade ribonucleic acid (RNA) molecules. It functions by catalyzing the hydrolysis of phosphodiester bonds in single-stranded RNA, thereby breaking down RNA into smaller fragments.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2 flow cytometer, by BD (2 mentions)
Modfit lt, by BD (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Facscalibur, by BD (1 mentions)
6 well plate, by Corning (1 mentions)
Ethanol, by Sangon (1 mentions)
Annexin 5 flous, by Roche (1 mentions)
Flowjo v 7, by FlowJo (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Facscanto 2, by BD (1 mentions)
7 protocols using rnase type 1 a
1
Cell Cycle Analysis of HSP70-siRNA Transfection
2
Cell Cycle Analysis after Hyperthermia
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After exposure to hyperthermia, the cells were re-incubated at 37°C for 0 h, 24 h, or 48 h. Then, the cell cycle phases were analyzed. Briefly, the cells were fixed with 70% ethanol at 4°C overnight. After washing with Ca2+-Mg2+-free Dulbecco's PBS, the cells were treated with 0.1 mg/ml RNase (Type I-A; Sigma, St Louis, MO, USA) and then stained with 100 µg/ml propidium iodide (PI; Sigma), in the dark, at room temperature for 20 min. After passing through a 40 nm nylon mesh, the samples were kept on ice until measurements. The data obtained, using the FACS calibrator, were used to analyze the cell cycle phase proportions with ModFit software. A cell fraction of DNA, below the sub-G0/G1 peak, indicated apoptotic cells; DNA histograms were used for their estimation.
For Annexin V staining, the cells were directly stained with PI and Annexin-V Flous (Roche) for 10 min and then washed with incubation buffer. The cells were identified with a FACS calibrator after setting the voltage using non-treated stained control cells. The cells were analyzed using Cell Quest software and classified into four different stages: unstained living cells, early age apoptotic cells stained only with Annexin-V, middle age apoptotic cells doubly stained, and late age apoptotic and necrotic cells stained with PI only.
For Annexin V staining, the cells were directly stained with PI and Annexin-V Flous (Roche) for 10 min and then washed with incubation buffer. The cells were identified with a FACS calibrator after setting the voltage using non-treated stained control cells. The cells were analyzed using Cell Quest software and classified into four different stages: unstained living cells, early age apoptotic cells stained only with Annexin-V, middle age apoptotic cells doubly stained, and late age apoptotic and necrotic cells stained with PI only.
3
Cell Cycle Analysis by Flow Cytometry
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Cell cycle analyses were carried out as follows: after treatment, cells were collected by scraping and centrifuging, and the cell pellet was resuspended with phosphate-buffered saline (PBS). The cell suspension was fixed with 1.8 mL of ethanol (70%) at 4 °C, for 1 h. Then, cells were washed twice by centrifugation, using PBS, resuspending the pellet in 300 µL of PBS containing 0.25 mg/mL RNAse (Type I-A, Sigma-Aldrich, Darmstadt, Germany) and 0.1 mg/mL PI. Samples were incubated in the dark at room temperature for 30 min. The PI fluorescence of 10,000 events was analyzed using a FACS Canto II flow cytometer (BD Biosciences, San Jose, CA, USA), with excitation at 488 nm and detection of the emission at 610 nm. Data were analyzed using the software FlowJo v. 7.6.2 (FlowJo, Ashland, OR, USA) [59 ,60 (link)].
4
Cell Cycle Analysis by Flow Cytometry
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Cell staining with the fluorescent propidium iodide (PI) was used to measure cell cycle distribution. The assays were carried out following the previous methodology described by Nicoletti et al. (1991). Briefly, 2.5 × 105 cells/well were seeded in 6-well tissue culture plates at 37 °C and 5% CO2. After 24 h of adherence, cells were treated during 48 h with hybrids 6e and 7 , together with the vehicle control. At the end of each treatment, cells were scraped, and the centrifuged pellet was resuspended in 1.8 mL of 70% ethanol at 4 °C to fix the cells overnight. Then, after centrifugation, cells were washed twice in versene buffer to completely remove the alcohol, besides, they were further resuspended in 300 µL of PBS containing 0.25 mg/mL RNAse (Type I-A, Sigma-Aldrich, Darmstadt, Germany) and 0.1 mg/mL PI. After the incubation in the dark (for 30 min at room temperature), 10,000 events were analyzed with a FACS Canto II flow cytometer and the software BD FACS Diva 6.1.3. (BD Biosciences, San Jose, CA, USA). The PI fluorescence signal was analyzed with excitation at 488 nm (using a Sapphire laser), and detection at 610 nm. The cell cycle model was fixed with FlowJo 7.6.2 (Ashland, OR, USA), applying the Dean-Jett-Fox model [43 (link)].
5
Cell Cycle Analysis by Flow Cytometry
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After transfection with the target plasmid or mock, phosphate-buffered saline (PBS) was used to wash the trypsinized cells twice, followed by cell-cycle phase analysis. This was done by fixing the cells overnight at 4 °C in 70% ethanol. Cells were washed with Ca2+/Mg2+-free Dulbecco’s PBS and treated with 1 mg/mL RNase (Type I-A; Sigma, St. Louis, MO, USA). Treated cells were then stained using 10 μg/mL propidium iodide (PI; Sigma) for 20 min; then, the cells were filtered and kept on ice until measurement. Cells were obtained using the FACS calibrator using nontreated stained cells. In this study, we quantified and took into consideration cell fractions with a DNA content lower than G0/G1, the sub-G0/G1peak.
6
Cell Cycle Analysis by Flow Cytometry
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Cell cycle analysis was evaluated by flow cytometry using propidium iodide (PI). This experiment was carried out as previously described by Nicoletti et al. (1991) [54 (link)]. Then, 48 h after treatment with the IC50 value of the hybrids 4a , 4g and DMSO (1%) as a vehicle control, cells were collected by scraping and centrifugation, resuspending the cell pellet in a versene buffer. Thereafter, cells were fixed with 1.8 mL cold ethanol (70%) at 4 °C overnight. Alcohol was removed by subsequent washing twice with versene buffer. The final pellet was resuspended in 300 µL of PBS with 0.25 mg/mL RNAse (Type I-A, Sigma-Aldrich, Germany) and 0.1 mg/mL PI and incubated for 30 min in the dark at room temperature. FACS Canto II flow cytometer (BD Biosciences, USA) was used to read the PI fluorescence of 10,000 events. The analysis of data was performed with the software FlowJo 7.6.2 (Ashland, OR, USA) [55 ].
7
Cell Cycle Analysis by Flow Cytometry
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Flow cytometry was used to assess the results of the cell cycle analyses using propidium iodide (PI),. Following a time of 48-h of treatment using DMSO (1%) as a vehicle control and the IC50 value of the D. viscosa extract. Cells were further collected by scraping and centrifugation, resuspending the cell pellet in versene buffer. Then, the fixation process with 1.8 mL 70% ethanol was carried out at 4°C for 1 h. After washing with versene buffer, the alcohol was eliminated. The final pellet was resuspended in 300 µL of PBS with 0.25 mg/mL RNAse (Type I-A, Sigma-Aldrich, Germany) and 0.1 mg/mL PI, and incubated at room temperature for 30 min in the dark. The PI fluorescence of 10,000 events was read using a FACS Canto II flow cytometer (BD Biosciences, United States). The software FlowJo 7.6.2 (Ashland, OR, United States) was used to analyze the data (Herrera et al., 2021 (link)).
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