Apotosis in RAW cells

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shakeelrm
shakeelrm's picture
Apotosis in RAW cells

Hiii
I am trying to look for Apotosis in RAW cells. I tried to do FACS analysis but it didn't worked as i have to scrap the cells and the cells usually die boz of it. I am now trying to shift to  immunofluorescence microscopy. If any body has suggestions please suggest.

Thanks
Shakeel

Chin Fen Teo
Chin Fen Teo's picture
 Hi Shakeelrm,

 Hi Shakeelrm,

An alternative method to scraping cells is to use EDTA as a cell dissociation reagent. You can purchase cell dissociation buffer from vendor (Gibco), but I found that using home-made 60 mM EDTA in PBS works just fine!

(I use this method to harvest cells for DNA fragmentation assay, which works superb!)

Briefly, you rinse cells once with PBS (warmed-up per usual), then add 60mM EDTA/PBS, incubate (37deg) for 5 to 10 min, and afterward you can collect the cells by washing off them the dish with ice-cold PBS.

Hope this helps solving your problem, and good luck.

shakeelrm
shakeelrm's picture
Thanks Pippuri for ur

Thanks Pippuri for ur Suggestion. Did you used adherent cells for ur assay.

Thanks
Shakeel

Chin Fen Teo
Chin Fen Teo's picture
 Hi Shakeelrm,

 Hi Shakeelrm,

Yes, my cells are adherent in nature. I have tried this method at least in 2 different adherent cell lines with great success. Never use RAW cells though. But I doubt it makes any difference...

Cheers.

samm
samm's picture
If you have lots of

If you have lots of conditions in a plate and want to assay 'untouched' cells, I'll recommend a combination of active-Casp3 (cells on plate - e.g. Promega Apo1 casp assay) + lactate dehydrogenase assay (from sup, to detect lysed cells; many vendors).
I've used Mediatech's cell dissociation buffer (trypsin free) on RAW and J774s - works fine for Annexin/PI and FLICA/PI for flow.
IF is not really quantitative, unless you can blind count 10+ fields/sample to get the ratios.

shakeelrm
shakeelrm's picture
hiii Samm

hiii Samm
Thanks a lot for ur suggestion. It is a quantitative assay that is why i want to doFACS analysis, and at the same time i am doing microscopy. I have done tunnel assay and it worked fine but there also Annexin/PI kit is not working. If you have any suggestions regarding whether i should fix the cells or shall i directly mount the coverslip on the slide.

Thanks

Shakeel
 
 

samm
samm's picture
There are several methods -

There are several methods - and I usually prefer multiple readouts in cell death studies.
For macs and T cells, a mix of the following works well from a publication standpoint.
IF: DAPI (total)+BrdU(TUNEL: DNA frag)+FLICA (active caspase)
FACS: Annexin/PI, cell cycle PI, FLICA, JC1 mitochondrial potential
Spectrometric/Fluorimetric 96well: use sups for lactate dehydogenase, and cells (lysate) for active caspase (Promega)
Best,
-sam