Apoptisis assay for adherent cells via flow cytometry

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Dals
Dals's picture
Apoptisis assay for adherent cells via flow cytometry

When performing apoptosis assay using annexin-V / PI  in flow cytometry........Whats the best way to harvest my adherernt cells so as not to damage membrane integrity (false positive annexin-v binding)...anyone has a detailed protocol??
 

 

 

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

 Hi Dals,

I use EDTA to harvest my cells for apoptosis assay.

Here is the steps:
1. Wash cells once with PBS (warmed).
2. Incubate cell with 60 mM EDTA/PBS, 37 deg for ~ 5 to 8 min (depends on your cells)
3. Wash cells off the plate with ice-cold PBS.
4. Spin down and subject to staining per usual.

Good luck.

marcus muench
marcus muench's picture
The answer depends a lot on

The answer depends a lot on how tightly bound your cells are to the plate.  Also the level of confluence affects recovery methods.  I often try to harvest adherent cells by scraping without the use of enzymes from cultures that are sub-confluent.  this helps preserve surface markers for flow that could be affected by trypsin. Other methods include the use of dissociation buffer (Invitrogen-probably contains EDTA and other goodies).  Dissociation buffer may take some time to work on really sticky cells.  You can also filter the cells to help remove clumped cells from analysis.  Doublet discrimination by flow cytometry also helps when your cells are clumpy to avoid counting clumped cells.

It is worth remembering that apoptosis and necrosis are different things and if you mechanically or enzymatically kill cells, they will be PI positive but not annexin-V positive.

samm
samm's picture
Hi, take a look at this link

Hi, take a look at this link for a general idea:
http://www.scientistsolutions.com/t2467-intracellular+staining+for+flow+cytometry+in+adherent+cells.html

Remember that the divalent cations in the annexin buffer can cause cell clumping, so you need to be careful.
I do not exactly know what your application is, but if you are only looking for early apoptosis (AnV+/PI-), go ahead and trypsinize your cells (monitor closely, and take care not to over-trypsinize). You'll get an increase in AnnV-/PI+ cells, but thats usually less than 5%; and at least for early apoptosis (driven by expt treatment), the benefits and convenience outweigh the risk of clumps and death caused by the slow detachment/mechanical disruption.

We have successfully used this for lung and kidney primary epithelial cells, and cell lines including MDCK.

Also, use an alternative assay such as Casp3 activation/LDH release (can be done in the same well: sups vs cells) for additional corroboration.