quantifying TUNEL: flow cytometry vs direct counting on slides? - (Oct/31/2007 )

Hi everyone, this is my first post, I'm so glad I discovered this forum!

I'm trying to find the best way to assess apoptosis with three different cell lines, they are all primary and adhesive. I tried annexinV/PI first, but I couldn't get them to be PI negative. I suspect I've been too rough on them during trypsinization and all the washing, and unintentionally damaged their membranes

So anyways, since that didn't work out so well, I decided to give TUNEL a try. After some paper-reading, I realized there are two ways to quantify TUNEL: either by flow cytometry, or using chamber slides or cytospin to adhere cells to a glass slide, performing TUNEL with an in-situ kit, and then counting by direct microscopy.

When I presented this discrepency to a senior lab member, he just gave me a cold stare and asked me which was more accurate: counting 400 cells or 10,000? However, I think there must be some reason why so many papers use the glass slide method !

Does it have something to do with the adhesiveness of cells (all the papers using glass slides use adhesive cells)? After all that failure and disappointment I went through with annexinV/PI, I don't want to make the same mistake again!

And another question: If I'm doing TUNEL via flow, is single staining enough or should I counter-stain it with PI or something? I'm not sure what the function of counter-staining in this situation actually is.... Anyways I'm a newbie in this apoptosis thing (my prof asked me to do this two months ago), any help at all would be greatly appreciated!

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Not an expert on this, but it seems to me that you should use flow cytometry if at all possible, however sometimes this is difficult with adherent cells etc which is why some people use the slide method, so it depends on your cells and if you can get the protocol to work for your cells... I think there is also an ELISA method that you may want to also consider.

Hope This Helps and Good Luck!

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Thanks for the reply! I ended up using FACS, and the results were great !

Another question: How much 1% paraformaldehyde is needed to fix 1X106 cells?

My protocol suggests 5ml paraformaldehyde for fixation and 5ml 70% ethanol for permealization. Can I cut them down to 1ml so I can do all this in eppendorfs instead of 12X75mm tubes? I always lose a bunch of cells with those tubes because of their round bottoms and my lousy pipetting

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