I am facing trouble in cell detachment with Saos-2 cells. I am using 0.25% trypsin and incubating it at 37°C for 10 mins and still the ells do not detach. I have increased the incubation time but to no avail.
hi,
if you want to split your cell I wouldn't use scrapper since its damage the cells you can use it only when you want extract a proteins from your cells. I would shaking very well instead before put your flask in the incubator and after, and I would use 5 minutes since 10 minutes so long it might be toxic to your cells and could activate other pathways which is something you might not want it.
good luck
Make sure that your trypsin solution is fresh and active. Also it should contain 0.03% EDTA as per ATCC protocol. Remove the culture medium from the flask and rinse with sterile saline or PBS to get rid of any residual serum. Serum will compete with the cells for trypsin will prevent cell detachment. Remove the PBS and add trypsin EDTA in PBS.
Trypsinize at room temperature or 37° C for 5 to 15 minutes with or without agitation. If trypsin EDTA does not work, instead try cell scraper. Scraping is a cruder method than trypsin EDTA, but it is a classical method used in cell culture.
Good luck!
http://www.atcc.org/products/all/HTB-85.aspx#culturemethod
Try to see if some other cell types are coming off with your trypsin and if not,
get the new bottle.
Hi Sarbani, I also think, as of Dr. Ishtiaq and Walter, check your trpsin EDTA solution - if it is okay or active. Best.
I have checked the trypsin-EDTA solution. It is working fine on other cells. And I do follow the procedure as mentioned in ATCC protocol. For cell splitting few cells are ok but for performing assays a large number of cells required. and here is the main problem.
In a few cases ,cells should be trypsinized before reaching high confluency(more than 80%).
Sarbani,
You indicate that routine, small scale transfers of these cells are not a problem, but when you scale up to grow many cells, the trypsinization problem occurs. If this is the case, you must consider the differences between the culture conditions in the 2 scenarios to identify the problem. Are the culture vessels identical or are you using small flasks for routine culture and large flasks or roller bottles for upscaled work? Is the starting cell number the same, do the cells reach the same level of confluency, are the cultures fed the same way? For many cell types, if they are grown to full confluency and then left that way for one or more days, they will become highly resistant to trypsinization. Are the culture media the same, are the volumes of tryp-EDTA used the same, etc. If you consider the culture conditions you are using, you should discover the problem.
Thanks Robert J Walter. Although the culture vesicles, starting cell numbers, and volume of tryp-EDTA is same, the confluency % is higher. May be the cells do become resistant when they are over confluent. I had never faced a problem of this sort before.
Wash twice with pbs after removing culture medium, and shake flask with some knocks after incubation
I have always used the brush head scrapper it will not destroy your cells if u scrape gently.
How confluent are your cells? I have found that if they are too confluent they take some time to come off. Try putting the trypsin on when it is cold and then it will get a chance to work its wall into to spaces between the cells, and then put it at 37C and they should disaggregate reasonably well.
Good luck.
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