We have been sorting the breast cancer cell line MB-231. However, the yield has been pretty miserable. As you may have experienced, MB-231 cells either become clumpy or sink to the bottom during the sorting process. I would like to ask if anyone has any ideas on how to better sort these cells. Really appreciate your help! Thanks in advance!
Try to dissociate your cells either with Enzyme-free cell dissociation buffer such as Gentle Cell Dissociation Reagent (Stem cell technologies) or Cell Dissociation Solution Non-enzymatic (Sigma) or Cell Dissociation Buffer, enzyme-free, Hank's Balanced Salt Solution (Life technologies) and then sort your cells.
The tips to overcome the trouble you have is to divide the samples for cell sorting as many as possible and not forget to suspend and pass through cell filter just before setting into the FACS machine. Tumor cell line is generally easy to form cell aggregation even under the suspended conditions, so that usage of trypsin-EDTA is recommended even if you sort them with antibodies against cell surface marker. For instance, CD44 is known to exhibit shedding after the treatment with trypsin. As far as I experience, however, the benefit to make them single cells is larger than the damage to the epitope.
I would spin cells down when preparing for FACS at lower speed and flick the pellet to ensure homogenous suspension of cells. Also, would definitely use a gentler dissociation agent than trypsin. You can add 1% FBS to the PBS that you resuspend your cells in to keep them viable and avoid clumping. This seems to have worked in a few lines that clump even faster than 231s. Additionally, you can improve yield by starting with a fairly high density so false negatives can be eliminated and your gain is higher.
Hi
We recently encounter the same problem. Have you gotten a solution to avoid the cell clumps ?
Thanks so much.
@Jitao Zhang, so sorry for the late reply! In the end, I settled for the usual trypsinisation method, after which I spin the cells down and replace the trypsin/medium with PBS + abit of FBS (2%). The cells have to be diluted quite a bit for sorting (for me, a confluent 10cm dish into 4-5ml of PBS + 2% FBS). The pressure on the sorting machine can actually be lowered to improve yield and lower abort rate.... needs a bit of tinkering.
Hi Dear MS CM
Thanks so much for this info, we also found that lower concentration would mitigate the problem some how. At high concentration, they still get cluster easily.
Thanks again and hope everything goes well.
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