Trypsin does not work - need to detach monocytes (Oct/11/2005 )
Hi, I was trying to trypsinize primary monocytes (extracted from the whole blood by density gradient Ficoll Pague centrifugation followed by 2-hour incubation at 37*C in order to remove lymphs from PBMCs). When I removed media and washed the attached cells with PBS, I added trypsin. After 10 min. only approx. 20% cells detached. Any suggestion? I really need to count the cells before RNA extraction!
Thanks in advance.
Paja
Pre-warm your trypsin to 37°C, wash your cells and extra time with PBS maybe?
How long have your cells been in this flask? I nottice that if you have your cells for too long in a flask, they are harder to remove (more attached to the flask maybe?).
How long have your cells been in this flask? I nottice that if you have your cells for too long in a flask, they are harder to remove (more attached to the flask maybe?).
Vairus,
Thanks for your replay. I am cultivating PBMCs for 2 hours to allow CD14+ to attach = separate them from the lymphs. According to some protocols even 24-hour incubation at 37*C is recommended, but based on some other ones, 2 hours should be sufficient.
I tried both RT-"warm" trypsin and trypsin pre-warmed at 37*C and I think I washed the cells enough to remove the media. Just to be safe first I added small amount of trypsin to splash the cells and removed it, and then I added the required amount of trypsin and let it act... (Without success.)
Strike the flask by hand, this will be helpful for detache.
Some tips:
You can try leaving the tripsyn a few more minutes.
Apart from warming the trypsin before adding it, you can place your flasks at the 37ºC bath while they are on treatment.
It is heplful to hit the flasks a bit as someone recommended you before and then resuspend the cells vigorously with a pippet washing the surface of the flask.
good luck,
sudaca
agree with everything sudaca says...
You may wish to try teflon coating flasks (they are expensive and few supplier make them). These flasks do not allow the monocytes to adhere to the bottom.
Hi Minnie Mouse
I need monocytes to adhere since this is a principle how to separate them from other PBMCs... But they attach too much to allow me to count them...
But thank you anyway - I had no idea there is something like teflon-coated flasks. Thanks
Paja
Hi Paja
I have the same problem as you did for detaching monocytes. I ahev triede trypsin and PBS EDTA solution. No sucess... have you managed to overcome the problem?
regards
you cannot use cell scraper?
I culture liver MSC or BM MSC, and when I need to pass the cell
I wash the cell with PBS, and add trypsin, leave few min
then tap the flask, and scrape the bottom to completely remove
cells.
I dont think that I am hurting cells.