Hi
I want to visually evaluate apoptosis in my transfected cells using annexin v-pi assay under fluorescent microscope...but my problem is that my transfected cells express RFP a red fluorescent protein which can intefere with red fluorescence of PI and cause false-positive results ...what should I do?
What about using flowcytometry instead of microscopy? Could this approach fix the problem?
TNX!
Yes Absolutely! Just substitute the PI with 7-AAD which has a far red fluorescence. You just have to run an unstained (i.e. RFP only) and single stained controls for the other dyes for compensation.
Technicaly is possible to change PI to another nuclear stain. First you have to prove the new stain usability. (Only important issue would be that the new stain should intake by only dead cells, if i remember well.) Likely a deep-red version of nuclear stain will be good.
Yes Absolutely! Just substitute the PI with 7-AAD which has a far red fluorescence. You just have to run an unstained (i.e. RFP only) and single stained controls for the other dyes for compensation.
You can get an Annexin V-FITC & 7-AAD kit from BD or any other brand. Culture cells on cell culture treated six well plates at a density of 300,000 cells per well. Treat cells with drugs for 6 hrs and then harvest the cells using trypsin/accutase.
Remember to set proper controls, Untreated cells, and cells treated with a standard like camptothecin for apoptosis induction. Also proper controls for compensation like single stained cells with annexin v FITC and 7-AAD and unstained to check for spill over from the RFP too.
After harvesting the cells wash them with PBS and follow the kit instructions. Briefly, resuspend the cells in 1X annexin v binding buffer at a concentration of 1 mil cells/ml. Take 100uL of cells and add 5 uL of ann v FITC to each tube. Incubate for 15min. Add the 7-AAD dye 5uL just before acquiring the samples. FL1 for FITC and FL3 for 7-AAD which will prevent interference from RFP after proper compensation.
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