Apoptosis

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Richie
Richie's picture
Apoptosis

Hi all,
I am here with a query.. I want to find whether apoptosis is taking place due to the agent i use on the meatazoan species. My problem is that i am not able to isolate DNA from it, and so, I need some way to prove apoptosis is taking place. I tried some stains, but they could not so easily penetrate the cuticular barrier for the parasite and hence cudnt differentiate live, apoptotic or necrotic cells.[i]
SO, can any body please suggest me with a solution???

Jitendra Sinha
Jitendra Sinha's picture
For checking if apoptosis or

For checking if apoptosis or necrosis is happening in your experimental set-up, you need to check the apoptotic markers available with various companies. You can use immunological assays or TUNEL also would be of some help to you.

A Timmer
A Timmer's picture
I would recommend using the

I would recommend using the TUNEL assay. Many companies make the kits and after trying about 3 different ones I concluded they are essentially the same. I found the BD Biosciences one to the be the cheapest (still really expensive though). The protocol involves fixing and permeabilizing the cells to allow the stain to enter and bind to the DNA. The assay is very quantitative by flow cytometry and can also be used for microscopy.

heehawmcduff
heehawmcduff's picture
I would also recommend

I would also recommend backing your TUNEL data up with at least one other type of apoptosis assay as it's been indicated in the literature that TUNEL can occasionally give false-positives.
Perhaps Western for caspase cleavage or Annexin V/PI staining

20101975
20101975's picture
The most commonly used assay

The most commonly used assay for apoptotic phenomenon is TUNEL but I have seen many false positve results which really make a mess out of your research so better to do some kind of ICC or ihc for caspaces like 3 and 9.

Shampa
Shampa's picture
Along with TUNEL Staining you

Along with TUNEL Staining you can also try the following:

1) Hoechst 33342 staining can be used to detect apoptotic changes.Hoechst 33342 is a vital DNA stain that binds preferentially to A-T base-pairs.

2) Annexin V + 7-AAD staining
Stages of programmed cell death can be enumerated using a dual parameter staining consisting of Annexin V-FITC and 7-AAD.

Annexin-V (-),7-AAD (+)   indicate Dead cells
Annexin-V (+),7-AAD (+)  indicate Late apoptotic/dead cells
Annexin-V (-),7-AAD (-)    indicate Live cells
Annexin-V (+),7-AAD (-)   indicate  Early apoptotic cells



 

Richie
Richie's picture
Thanks for your reply.. my

Thanks for your reply.. my problem is i am checking the process in whole organism as a nematode, so if i go for staining protocols, as i tried with EB/AO staining, i could get some fluorescence, but i am not sure whether it was due to cell death, like whether it could actually penetrate the cuticular barrier, and in case i use TUNEL assay, i dont have access to Flow cytometry, so is their some other way out..
Awaiting some guidance from your expertise to solve my problem.

heehawmcduff
heehawmcduff's picture
Hi

Hi

Rather than staining your organism and looking for morphological indicators of apoptosis, it may be worth performing a protein extraction and running a western blot for biochemical indicators of apoptotic processes such as cleavage of caspases.