That is the point, to shock the cells so they "release" (basically so they start retracting into a more spherical shape rahter then the more spread out morphology).

The only time we use 37oC is if we use trypsin, but this cause issues if you are looking at adhesion molecules...

For most purposes simply scraping the cells from the plate should be fine. For specula purposes we use accutase, which we use at RT with shaking (20-30 min).

Hi Stéphanie,

If you only want to detach the macrophages from the wells you can use 1x trypsin and incubate the plates at 37 degree for 10-15 min. I prefer to use TrypLE from company called Gibco, it also act in similar fashion as trypsin. If you wish to isolate RNA from macrophages best is to use cell lysis buffer provided with the kits. Now coming to your question, Why you use EDTA? because EDTA is a calcium chelator that will remove the Ca2+ ions that integrins require to maintain cell adhesion. Intigrins are really a important player in maintaining cell to cell and cell to surface attachment and also for their activity intigrins need Ca2+ ions. Once Ca2+ ions are captured by the EDTA, intigrins can not function normally and therefore cells get loosely attached and can easily be detached by washing.

I assume you must be knowing that PBS is phosphate buffer which keeps osmolarity and pH constant thus causes no harm to the cells and thats ehy we use PBS in cells washing or in staining etc.

Coming to the point of temperature shock, I would also be shocked if someone transfers cells directly from 37 to 4 °C. However, it also depends on experiment you are planning to do (such as to fridge the enzyme activity or amount measure may be you need cold temp) but in general just for detaching the cells, transferring them directly from 37 to 4 °C is above my imagination. Good luck

Hi Dhirendra,

that is exactly my point. We cultivate them at 37°C, detach them at 4°C with the PBS-EDTA, to seed them and cultivate them again at 37°C and do infection on them. For the use of EDTA, I was also using (in trypsin) for my keratinocytes (H. Green protocol), so I'm not shocked at all. That's just the temperature shock which is, for me, problematic. I think I will do a trial of detachment at 37°C and 4°C and compare the death rate between the both conditions. Because I'm really annoyed by the temperature break. For the RNA or the proteins, as you said, I do direct RNA lysis buffer or 4°C PBS quick wash before protein extraction.

Anyway, thanks to all for your replies

Yeah try it and let me know the results if possible, I am really curious to know. Good luck.