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24 h experiment to detect live cells, no additional treatment which dye best to use? Draq5 (for live cells) - too toxic Draq7 is for staining dead and permeabilized cells
Asked on Aug 03 2011
Thank you for contacting us. I have received the following response from the lab: We would suggest to approach this as follows ... Treat the cell culture with 1-3 uM DRAQ7 during the incubation, image to enumerate the dead/leaky cells at the required end-point and then immediately add 3-5 uM DRAQ5, allow to stain for 5 -10 min and measure again for ALL events, IN THE SAME CHANNEL. The two images will show: 1) dead/apoptotic cells, DRAQ7+ 2) all nucleated cells, DRAQ5+ The two images can be artificially colored to reveal the live cells, when merged. The DRAQ7 positive events can be monitored by imaging in real time during the culture period to observe the progression and dynamics of cell damage and death. Red excitation will limit the risk of DNA damage that is common to UV excited probes. The Draq5 would need being added at the very end of the 24 hour time point, after the last DRAQ7 read. It is tricky to measure the live cells at the beginning of the experiment (i.e. around 0 or 1 hour). We would consider using phase contrast to estimate total cells and subtract the initial DRAQ7 positives. There is no real way to remove Draq5 after incubation for 10 min or so. Once it’s labelled to DNA then it’s there “irreversibly” due to high affinity. As Draq7 is not cytotoxic, an incubation with Draq7 for 24 hours would be fine. DRAQ7 only enters cells that are membrane-compromised. Our data show effectively no toxicity after 96 hours of proximity to proliferative cells. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
Answered on Aug 03 2011