Hi Sarah!

You  can use Annexin V/PI either with flowcytometry or fluorescent microscopy to discriminate early apoptosis from late apoptosis/necrosis based on the translocation of phosphatidylserine to the outer layer of the plasma membrane of early apoptotic cells which is stained by Annexin V-FITC and the DNA which is stained by PI in the late apoptotic/necrotic cells.

The description of apoptosis, late apoptosis/necrosis is as follow:

"Ann+/PI- early apoptosis

Ann+/PI+ late apoptosis

Ann-/PI- live cells

Ann-/PI+ necrosis”

You can also use Rhodamine 123 which is a green fluorescent dye for apoptosis test.

Maybe this file is useful for you.

http://www.bioscience.co.uk/userfiles/pdf/Apoptosis_Necrosis_and_Cell_Viability_Assays.pdf.

Western blotting (like PARP or caspase 3 cleavage), DNA fragmentation assay and FACS would also be recommended to know what you actually observe.

Also you can use Real Time PCR for your genes of interest to find the extra pathway of the apoptosis in addition to the anti/pro apoptotic genes. 

Hope you can cope with your test.

I prefer to you to go with conventional gold standard fluorescent staining and microscopy to qualitatively analyse positivity for apoptosis. Followed by that you can perform quantitative assays like TUNEL assay (kit based). Further, to study caspases you can perform western blotting/flow cytometry (FLICA/caspase-specific tagged antibodies) which is very specific.

Thanks Naveen, the only problem I have with section staining is that I want to look within the stem cell-enriched population, hence would sort cells or gate cells for stem cell markers. Unfortunately I can't find any conjugated anti-mouse antibodies against active caspase-3! So I think FLICA is the best approach there. 

Yes. And apart from that if you want to check levels of active caspase-3 particularly, you can use caspGLOW red active caspase-3 staining kit for flow cytometry from BIOVISION. It stains for active caspase-3 in live cells and it is non toxic.

We use primary smooth muscle cells line and we use TUNEL or AnnexinV by immunofluorescence, but they both measure cell death, not specifically apoptosis.

Other than pro-apoptotic proteins by western blot, we are also starting to use "Magic Red" which measure the caspase activity: http://www.immunochemistry.com/products/caspase-3-substrate-magic-red-caspase-3-assay-kit.html

Good luck!

Hi Sarah!

You  can use Annexin V/PI either with flowcytometry or fluorescent microscopy to discriminate early apoptosis from late apoptosis/necrosis based on the translocation of phosphatidylserine to the outer layer of the plasma membrane of early apoptotic cells which is stained by Annexin V-FITC and the DNA which is stained by PI in the late apoptotic/necrotic cells.

The description of apoptosis, late apoptosis/necrosis is as follow:

"Ann+/PI- early apoptosis

Ann+/PI+ late apoptosis

Ann-/PI- live cells

Ann-/PI+ necrosis”

You can also use Rhodamine 123 which is a green fluorescent dye for apoptosis test.

Maybe this file is useful for you.

http://www.bioscience.co.uk/userfiles/pdf/Apoptosis_Necrosis_and_Cell_Viability_Assays.pdf.

Western blotting (like PARP or caspase 3 cleavage), DNA fragmentation assay and FACS would also be recommended to know what you actually observe.

Also you can use Real Time PCR for your genes of interest to find the extra pathway of the apoptosis in addition to the anti/pro apoptotic genes. 

Hope you can cope with your test.

You can use luminometric assays that can measure initiator and executioner caspases (Caspase-Glo assays).  You can also use a superarray (Qiagen) to measure up/down regulated apoptosis genes.

Since you want to look at a subpopulation of cells, you might want try TUNEL based flow cytometry combined with stem cell markers, which can also used for microscopy. TUNEL however only measures cleaved DNA and positive cells are not necessarily apoptotic. FLICA has the disadvantage of not being specific for a given Caspase, but shows the activation of Caspases. Annexin V has problems with adherent cells because of trypsinization of adherent cells can give false positives - Invitrogen I believe has an alternative to Annexin V. You always have to use two methods, but in your case you either have to sort cells for a WB and/or do flow using markers to gate your cells and use TUNEL, FLICA. By the way, with the latter approach you might also detect bystander apoptosis, which might lead some other results.....

Also look at this product

http://www.lifetechnologies.com/order/catalog/product/V35114?ICID=search-product

and at these reviews

Apoptosis and Beyond: Cytometry in Studies of Programmed Cell Death

Donald Wlodkowic,* William Telford,† Joanna Skommer,‡ and Zbigniew Darzynkiewicz 

Beyond annexin V: fluorescence response of cellular membranes to apoptosis

Alexander P. Demchenko

Hi Sara,

For viability studies, I usually performed microplate assays, for instance MTT (http://www.ncbi.nlm.nih.gov/books/NBK144065/) or LDH quantification assays (http://www.piercenet.com/product/ldh-cytotoxicity-assay-kit). However, these assays measure cell metabolism and activity, then you assuming that dead/apoptotic cells show lower absorbance (MTT) or higher absorbance (LDH) than control/alive cells.

For direct apoptosis measure of your cell population, I think a good method is Annexin-V / PI staining. Annexin-V shows you the cell population which are exposing phosphatidylserine to the outer membrane, that means the cell is undergoing apoptosis. PI shows you the percentage of the cell population is directly dead. Combining both stainings, you should be able to calculate the percentage of early apoptosis (AnnV+/PI-) - late apoptosis (AnnV+/PI+) - dead (AnnV-/PI+) populations of your sample, acquiring data by flow cytometry.  I think is a relatively easy and cheap method, I hope it helps.

Good luck!

Jordi

Tunnel assay and Prpidium iodide assay are more reliable. But you can do it by WB and PCR as well.

Good luck

I used western blotting for PARP cleavage assay.

Thank you all so much for your suggestions and input, think I'll have to go away and research all these methods and try a few more things/kits. Hopefully I can get it to work!

There was recently an 'Science' webinar on the subject:

  http://webinar.sciencemag.org/webinar/archive/many-roads-cell-death

Hope it's useful for you. Best regards. Salut †                                        

Hi Sarah, you can use flow cytometry to look at Annexin V (apoptosis), SYTOX (necrosis / dead cells) and C12 resofurin (viable cells). Also PI / Hoescht can be used to stain live cells, and manual cell counts can be made. In addition, you can look at mitochondrial membrane potential using JC-1. Mitochondrial membrane potential will be decreased in early apoptotic cells. This can be used by FACS or conventional fluoresence microscopy. You could also try Western blotting for cleaved Lamin A, also a marker of apoptosis. Good luck!

Keep in mind that apoptosis is an on-going process and that certain tests are time specific for instance when you have Annexin V positive cells you have already lost your robust Cleaved Caspase 3 signal. When you have PARP cleavage you are too early for other markers. What we have done in the past is side by side time-courses in drug treatments doing western blots for PARP and Flow for Annexin V/DAPI and Cleaved Caspase 3. This works fabulously since you get a good idea of kinetics for apoptosis when all are combined.

The best method to study apoptosis in vitro is using MTT test please see my article, Khorshid F.A., J.S. Alameri. Apoptosis Study on the Effect of PMF on Different Cancer Cells, International Journal of Biological Chemistry,2011.