Keeping apoptotic/detached cells for WB? - (Jun/05/2014 )

Hi!

I'm pretty new to this so feel free to laugh if the question is stupid, but please provide advice if so :)

I have been transfecting cells with an apoptosis inducing gene, and a bunch of them detach from this treatment. Now I want to analyze protein levels in cells treated with this gene. Should I discard the floating cells and only use the cells that are still attached, or should I also include the floating cells?

I guess the apoptotic/floating cells could be leaking and they will have protein levels and gradients different from healthy cells, so maybe one should not include them? But then again, I want to look at protein levels in cells that were succesfully transfected. The cells that are still attached might be non-transfected cells and cells that for some reason are "immune"?

Advice much appreciated.

Those cells that detach are likely to be dead (you could test this - collect some and trypan blue stain), thus the proteins are probably in the process of being degraded.  The reasons why they have died could be either an effect of the transfection alone (i.e. not due to the gene, but rather the process itself - make sure you have appropriate controls) or due to the gene - this is unlikely before about 12 hours.  Note that you can try a range of different transfection methods and reagents to find the one that gives you the least death from the process.  In my hands the lipofectamine series (apart from RNAiMax) are all quite toxic, but the fugene ones are very good.

The cells that remain attached will be a mixed population of transfected and non-transfected cells, but unless you have added a marker to your gene, you won't be able to tell which is which - you could try FACS if you want to select just the population of cells transfected.  Liposphere based methods such as Fugene or lipofectamine will typically give less than 30% transfection. 

I would personally discard the cells, especially if they only make up a small proportion of the total cells.

Thanks for your reply bob1!

The plasmid has a fluorescent protein, and approximating from fluorescence microscopy, some 30% or so of the cells seem to express this marker. I also noted that most of the floating cells exhibited a lot of strong fluorescence, but maybe they have different spectral properties, at least in part, because they are (probably) apoptotic... But this, and the fact that I didn't notice them until 48hrs post-transfection leads me to believe that they underwent apoptosis from expression of protein rather than from the transfection procedure as such.

The floating cells made up like 15% (approximation), but I didn't make any thorough quantitative comparison to the controls. Trypan blue staining was a good idea to assess their status, I should have thought of that. Maybe next time I should make a proper count of trypan blue stained floating cells and make a comparison between gene-exposed and control samples.

 I didn't have duplicates for all conditions, but in the future, I'll try both with and without floating cells to see if it makes any difference. But like you mentioned, I guess it shouldn't make a very big difference as long as the floating cells don't make up a large part of the cells.

Follow up question (tad bit off topic maybe); what if I wanted to assess % apoptotic cells in drug exposed vs non-exposed cells by some apoptosis stain. Then I'd think the floating cells should be included?

Maybe it is hard to tell, but in general, would you say that apoptotic cells often detach to a large extent? It could of course vary much between cell lines and conditions. From literature I get the impression that apoptotic cells tend do detach a lot, but that apoptotic cells can also stay attached. Maybe this varies with time/stage of apoptosis, but I didn't find a lot of info on time frame for detachment of apoptotic cells. Does anyone have any idea?

As you say, it depends - some cells detach, some don't.  If you look at any given attached population of many cell lines you can usually find a few that are exhibiting the characteristics of apoptosis, even fairly late stage (blebbing), but are still attached.  Having said that, if you look at the floating cells, you will likely see some floating that are in the same stage as well. 

For your stain experiment, I would be tempted to detach them all and keep the floaters and run them through a flow cytometer if possible (needs fluorescent dye) - it would probably give you the most accurate data.  I would probably do a time course associated with this to see the relative timing of the apoptosis events, which will help you with your future experiments.