Transwell and apoptosis

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KatarinaLy
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Transwell and apoptosis

Hi!
Have any of  you used the transwell system for cytotoxicity testing? If so what method did you use to measure apoptosis in the insert?
Katarina

heehawmcduff
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Hi Katarina,

Hi Katarina,

I have used the transwell system for cell culture and apoptosis studies.  In your case, it really depends if you want to detect apoptosis in situ, in which case immunohistochemistry for a classical apoptosis marker such as cleaved caspase-3 in combination with Tunel staining or perhaps Hoescht staining to look at nuclear morphology would be a good starting point initially.

I would suspect that due to the limited size of the transwell and number of cells available it would be more difficult to extract protein for blotting or to remove cells from the surface for Annexin V/PI staining.

samm
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Hi! The FLICA system with

Hi! The FLICA system with DAPI works great for IF with the polycarb transwells, down to the 24 well size. If you use the 96 well inserts or are really limiting in cell numbers or have too many conditions, I'd recommend a fluorimetric read with something like Promega's CaspaseOne/Caspase Glo (something of the sort - essentially similar to FLICA in that it uses a active Casp3 inh coupled to a fluorimetric dye).

KatarinaLy
KatarinaLy's picture
Hi Samm!

Hi Samm!
The promega system seam perfect since I intend to use the 96 well plates and have a LOT of conditions to test. Do you think it is possible to ad the substrat direcly in the insert and measure? Or will I have to take them out? If it would be possible to do the readout in the transwell plate that would be so much easier!!
tanks for all your help!!

samm
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Hi Katarina! You can remove

Hi Katarina! You can remove the bottom media (you might be able to use this for an LDH release assay in 96 well plates - just to get another readout), freeze down your entire plate, then thaw, add the dissociation/staining buffer on the transwell, spin and read the plate. Just to be on the safe side, I'd recommend a top and bottom read with the transwell inserts in place, followed by another read without the inserts.