I am trying to lyse endothelial cells grown on 24 well transwell filters with a culture surface of 0.336 cm2. I am aware that I will have to pool some transwells in order to get a decent amout of protein. However, somehow I cannot get the cells from the filters. Does anyone have any tricks or a good-working protocol?
Scraping the surface into your chosen buffer or lysis reagent will be the answer. A rubber policeman would be best but is probably too large for the wells you are using so you could use the pipette tip you are using to aspirate the well. Add your reagent by pipette, use the same pipette to then scrape and aspirate the lysed cells. You could then use the same tip if you are combining wells or change tip for each well.
Yes, a rubber policemen is too large as the 24 transwell filters correspont to 96well surface area. I will try scrape of the cells according to Jerry Alan Warden-Smith's suggestentions.However, I already tried it like this but did not have a cell pellet in the end. Maybe I just need some practise! ;)
I wouldn't necessarily expect a pellet, the amount of material will be small, just assume the pellet is there and extract what you are looking for anyway.
Pipette tip works well but maybe for proteins you should make a pool. I obtained good and enough RNA from 24 transwells (endothelial cells) scraping with the tips, but never done for proteins. It also depends on what you want to do with the proteins. Be careful when using the tips. The first time I destroyed the transwell :)
Using pipette tip for scraping is better, but gently and aspirate them immediately after adding lysis buffer. I will also suggest you do this in cold condition, on ice or/and in cold room preferably because of the integrity of the protein release after lysis. Please do not expect pellet as such because of the low surface area of the transwell used and your protein will be little. So think about the quantity of protein you need. If quantity is a question then try using flasks (T25, T75 etc) to culture the cells you will have larger protein
I'm not sure if this exact size will work for a 24 well plate, but I have used this on a 6 well transwell dish and it works really nicely. (Falcon Cell scraper) https://catalog2.corning.com/LifeSciences/en-US/Shopping/ProductDetails.aspx?categoryname=&productid=353086(Lifesciences)
Conversely, you could cut the filters out with a razor blade and immerse in lysis buffer (even less than 1 mL could work) and you may get some lysis of cells.
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