Hi colleagues,
I have a problem with my sorting cells.
I isolated human keratinocytes from epidermal skin and I mark them with CD44 and ALDH to sort stem cells from them.
The time of staining takes around 3 to 4 hours and then I took them to the flow cytometer to sort the positive cells from negative ones with a sample pressure between 4 and 7, and this step took around 2 hours, then I returned back to my lab, do my calculations, count the cells to be plated (around 30 min) and plate the cells with media. Finally I incubated them. I changed the media every other day, but for couples of weeks they didn't form colonies compared with the non sorted cells which might start to form colonies 4 days post incubation.
Does anyone has any tips how to improve the cells viability or why they didn't show activity like the non sorted cells?
Is there any thing I should do to get more healthy or active cells?
Is that from the sorter?
Does anyone has the same situation?
Note: I repeated this experiment couples of times using neonatal, adult and aged skin.
I still have cells 2 months in the incubator now, they are not contaminated, I could say I could see some few divisions but zero colonies.
Hello Ayman Khalifa
You could use serum or protein in your buffers as this will increase viability of your cells. Use 1-3% BSA or FBS in buffers. It will help keep the cells healthy and viable without making them too sticky. You can also coat your collection tubes with protein prior to sorting as this will help reduce the chance of sorted cells from sticking to the sides of the tube. This will increase sort yield and cellular recovery.
Also, use the right temperature. Keep cells on ice but not for too long. Keeping the cells on ice can preserve viability. However, cold temperature can also prevent cells from repairing sort-induced damage. So, the longer the sort, the more damage done to the cells which cannot be repaired.
Hope this helps.
Best.
Thanks Malcolm, I will put your notes in my consideration in upcoming trials.
So usually I used fresh media in collecting and sample tubes, do you recommend to use buffer instead? Also I never coat the collecting tubes before, but I tried to use more media in the tubes hoping the cells not stick to a dry sides of the tubes during sorting, is the 0.1% collagen will be good to coat the tube?
thank you
You can use a concentrated media that has a high FBS content (~20%) to help cells recover from the sorting process. You can coat the tubes with 1% BSA. Do not use collagen.
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