As previously described (Turn et al., 2020 (link)), unsynchronized cells were prepared for flow cytometry by trypsinizing the cells, taking up the cells in ice-cold phosphate-buffered saline (PBS), washing with ice-cold PBS, and fixing the cells with ice-cold 70% ethanol dropwise while simultaneously vortexing to reduce the risk of cell clumping. Both supernatant and adherent cells were collected to ensure that we had a full representation of the cell population. The day of flow cytometry, cells were spun down, washed with ice-cold phosphate citrate buffer (0.1 M citric acid in PBS, pH 7.8) two times, treated with RNase A for 15 min (100 µg/ml; Sigma; R5125), and treated with propidium iodide for 45 min (50 µg/ml; Sigma; P4170) to stain for DNA content. Cells were passed through a cell strainer and run on a FACSymphony A3. The G1 peak of WT cells was set at a 50,000 voltage for each run, and these settings were used to acquire all subsequent samples run that day to ensure that we accurately track 2N, 4N, and >4N peaks. At least 10,000 cells were collected per sample. FloJo software was used to plot data shown.
R5125
Manufactured by Merck Group
The R5125 is a laboratory instrument designed for DNA extraction and purification. It utilizes a centrifugal mechanism to separate and isolate nucleic acids from various sample types. The core function of the R5125 is to provide a reliable and efficient method for extracting high-quality DNA for use in downstream applications, such as PCR and genomic analysis.
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Lab products found in correlation
2 protocols using r5125
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Cell Cycle Analysis by Flow Cytometry
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Cell Cycle Analysis by Flow Cytometry
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MCF7 cell 1x105/well/6well plate was cultured overnight before treatment with the various doses of inhibitors. After 24h, cells were harvested and washed with cold PBS before adding 5ml 70% ethanol. Samples were kept at 4°C overnight, and then they were centrifuged at 850g for 5 min and washed with cold PBS 2 times. 500μl RNase (R-5125, Sigma, 2mg/ml) and PI solution (P-4170, Sigma, 0.1mg/ml in 0.6% Triton X-100) were added and kept in the dark for another 45min before the FACS analysis.
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