i am culturing calverial cells from 3day old rat pups ,i seeded in 25cm corning culture flask. when i gave 0.25% trypsin treatment they are not at all detaching from flask even after 20 min....so pleas can any body healp me in this regard
Hi,
you have partial detachment so your trypsin is working: did you properly wash the adhering cells with buffer like PBS (for at least 30 s) to remove proteins from the medium, which may inhibit trypsin action ?
After removing media wash properly with 2mL of PBS or DPBS and trypsinize with 2-3mL trypsin till cells come off (Observe under microscope)
hellow sirs thank u very much for ur responcee...i did that step too,this problem is only with corning culture flasks,this situation is not with nunc and other culture flasks
Use first PBS twice, then EDTA (2 mM final concentration – 2 min) alone, then trypsin EDTA (5 min 37°C). If it doesn’t work use then mechanic shock (pat the side of petri dish).
Why don't you just move back to the nunc or other culture flasks? The other product you could try is accutase. It's a lot gentler on the cells, and you can leave it on for a long time without causing cell death. You still have to wash with warm PBS, but I've managed to use that product to get stubborn cells to detach. Hope you find a solution!
I suspect your problem is due to the charge on the plastic used to make the culture flask. We noted that when empirically testing flasks from different manufacturers for culturing our cells. Our method around the charge issue was to coat the flasks with 1% type-I collagen. In that respect the cells would attach to the collagen instead of the plastic through two binding sites, a calcium-dependent binding site and a RGD-dependent binding site. One of the major problems with using trypsin as the releasing enzyme is that it will also "punch holes" in cell membranes, cleaving cell membrane proteins at lysine-arginine sites. This gives one a myriad of dead and dying cells after enzymatic release. We circumvented that problem by releasing the cells with collagenase rather than trypsin, which would cleave the substrate (type-I collagen) without harming the cells.
Also, EDTA chelates divalent cations in general, i.e., calcium, magnesium, zinc, copper, to name a few. Depending on what you want to do with your cells post release, one or more of those divalent cations that are not calcium can be very important to the outcome of your experiment. Such was the case with our cells. Thus instead of using EDTA we switched to using EGTA, which is a specific chelator for calcium. It did not effect the outcome of our experiments when other divalent cations were necessary for functioning of the cells.
Our release procedure was to wash twice in DPBS only, once in DPBS-EGTA (watch cells round up), followed by collagenase-DPBS-EGTA (watch cells detach from flask). The cell suspensions were then added to 1% collagen in DPSB to negate the activity of the collagenase enzyme, centrifuged, the supernatant decanted, the cell pellet resuspended and then used for whatever experiment we had in mind.
Did you try scraping the cells with a cell scraper? This is a common technique used to gently remove stubborn cells from culture flasks/plates/dish after trypsinisation for 5 mins. It is a very effective method.
thank you very much for all of you for your valuble suggisitions,i will try them
Just a small insight based on my own experience with these cell types: instead of washing with PBS, I wash quickly (~ 10 secs) with a small volume of 0.25% trypsin to "pre-treat" the cells for detachment. I remove this, and add a second volume of 0.25% trypsin and incubate. The cells usually begin detaching almost immediately and are completey (or close to completely) detached after 3 mins. I also agree with Gayathri, for really stubborn cells, a *light* scraping can really help.
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