Hoechst stain

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Avene
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Hoechst stain

I am doing Hoechst stain to detect apoptosis in my cells. Apoptotic cells have condensed chromatin which would be stained brighter than that of normal healthy cells. However, when cells undergo mitosis, their chromatin also condensed, So how do we differentiate the condensation and more brightly stained chromatin between apoptotic or mitotic cells? Flow cytometry assays also show that about half of the cell population are in G2/M arrest. so, in this case, is Hoechst staining a good way to detect apoptosis?

samm
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Sadly, Hoechst staining

Sadly, Hoechst staining remains a rather subjective and non-quantitative staining / apoptosis assay. If you do not have any cell number and time limitations- which will be true of most cell lines(adherent or suspension) and some primary cultures too, your best bet is to have the cell cycle PI staining in conjunction with non-permeant-PI/Annexin-FITC in a FACS assay (or similar combination - there are 4 popular combinations of Annexin conjugate/late apoptosis/necrosis markers available that I know of).
The advantage of Flow cytometry is that its both objective and quantitative- which is very important for cell death assays, plus it deals with single cells in a population.

Sum
Sum's picture
samm wrote:best bet is to

samm wrote:

best bet is to have the cell cycle PI staining in conjunction with non-permeant-PI/Annexin-FITC in a FACS assay (or similar combination - there are 4 popular combinations of Annexin conjugate/late apoptosis/necrosis markers available that I know of).

Can you tell me what late apoptosis markers are available that I can use ?
Also, apart from FACS-dependant techniques, in what other ways can I quantify apoptosis % ? ( I am working with Peripheral Blood lymphocytes exposed to UV radiation.)

samm
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Apoptosis stage markers in

Apoptosis stage markers in addition to annexin/PI will include permeabilized PI (FACS) or EtBr (Agarose gel EP) for DNA fragmentation,
mitochondrial potential transformations with JC-1 (confocal/fl microscopy and FACS),
PARP cleavage and caspase activation (lots of colorimetric/fluorometric/ELISA/IHC/FACS kits are available, or you can just get the Abs and set up your own assay),
the FLICA/FLIVO system (do a search for the company here on Solutions search - above)
lactate dehydrogenase release (sometimes crude, but works well for some cell types: did not work very well for T cells in my lab)
etc.
Other than LDH, all the rest have worked well for me with PBMCs/purified T cells.
Hope this helps!
-sam

Sum
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thank you !

thank you !

Cell biologist
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Hi

Hi
I am also looking for a good merker for apoptotic/ dead cell by immunofluorescence. I used annexin V for FACS analysis. Now I want to study in fixed cell for immunofluorescence imaging, my transfection vector is GFP tagged, so please suggest something that will not ineterfere with GFP.
Thanks
 

samm
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Cell biologist wrote:

Cell biologist wrote:

Hi
I am also looking for a good merker for apoptotic/ dead cell by immunofluorescence. I used annexin V for FACS analysis. Now I want to study in fixed cell for immunofluorescence imaging, my transfection vector is GFP tagged, so please suggest something that will not ineterfere with GFP.
Thanks
 

 
You can try doing a TUNEL assay - use anti-GFP-FITC/AF488 to ID cells (just in case the GFP dies), and anti-BrdU-biotin::streptavidin-AF633/APC (assuming your FACS machine has a red laser at 633 nm) to assay DNA fragmentation.

heehawmcduff
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You could also do Western

You could also do Western blotting for caspase-3 cleavage (apoptosis) or caspase-1 (pyroptosis)

samm
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heehawmcduff wrote:You could

heehawmcduff wrote:

You could also do Western blotting for caspase-3 cleavage (apoptosis) or caspase-1 (pyroptosis)

 
Good point - except scratch the Western bit (I'm guessing the switch from flow to IF is to obtain localization/morphology data) : I stained for TUNEL-FITC, Cleaved Caspase 3 Cy3 and DAPI last year - worked out well, and you don't need special filters on your 'scope. If you have a 'scope with a red laser, try finding red shifted secondary/tertiary reagents (AF633) to minimize autofl.

samm
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samm wrote:

samm wrote:

heehawmcduff wrote:
You could also do Western blotting for caspase-3 cleavage (apoptosis) or caspase-1 (pyroptosis)

 
Good point - except scratch the Western bit (I'm guessing the switch from flow to IF is to obtain localization/morphology data) : I stained for TUNEL-FITC, Cleaved Caspase 3 Cy3 and DAPI last year - worked out well, and you don't need special filters on your 'scope. If you have a 'scope with a red laser, try finding red shifted secondary/tertiary reagents (AF633) to minimize autofl. The FLICA Red system works pretty well in that regard (non-Ab based active caspase 3, very low autofl)

michlyn
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Hello,

Hello,

I am looking for a good assay to use for apoptotic cell death in primary neuron cultures from mice. Suggestions?