Does anyone know how to reduce cell clumping after cells are treated with enzyme-free dissociation buffer? Is autoMACs rinsing solution (contains PBS and EDTA) helpful to prevent cell clumping in cell suspension?
Any methods are useful to make a better single cell suspension?
You may need DNaseI to prevent clumping! the clumping caused by DNA release from death cells. In my case, I isolate the single cell suspension from tumor mass by using this protocol:
Tumors were collected, minced using a scissors, incubated with 1 mg/ml collagenase D and 50 ug/ml DNAse I (Roche) in PBS for 30 min at 37°C, and filtered through 70-mm cell strainers.
A similar question was recently adressed here:
https://www.researchgate.net/post/How_can_I_separate_the_cells_of_my_PC-12_cell_line
The buffer might help, especially if it's calcium free. However, cell clumps are typically effectively dealt with mechanically via trituration. Essentially pipette them up and down several times and allow the fluid forces to break up the clumps. If you're already doing that and it's not working, try a small gauge needle instead. Transfer the suspension into a sterile disposable syringe, cap with a 22 or smaller needle, depending on the size of the clumps, and gently expel the solution. Repeat a few times, as needed.
Hi guys,
I have the same problem after the dissociation of canine mammary tumors. Peter, I've tried all you said, pipetting up and down, needles, trypsin, everything.. But the clumps are still there. They are small clumps, 5 to 10 cells, but when you need a single-cell suspension is really annoying. My last hope is DNAse I!
Hi all,
I have a similar problem!
I am dissociating adult mouse brain and then do FACS sorting with subsequent RNA analysis.
My cells clump a lot and I want to find a way to prevent that. Also I notice that clumping increases with time and also a lot when I centrifuge the cells.
The thing is that I am doing everything at 4 degrees (even the enzymatic dissociation) and I don't think DNAse will help, since it's not active at 4 degrees.
I use Calcium- and Magnesium- free buffers and also add EDTA (0,5 mM).
What else do you think would help to prevent clumping?
Thanks!
Nefeli Kakava may you share your protocol using Calcium- and Magnesium- free buffers and EDTA (0,5 mM)
Thank you
This was my latest protocol, but the optimized it more after I stopped working there:
https://www.biorxiv.org/content/10.1101/2020.07.23.217729v2