Greetings,
I am currently sorting a rare population of cells by flowcytometry. Getting very small amount of the sorted cells in a volume of 5 ml (including sheath buffer and 2%FBS/PBS). Tried so hard to make a pellet from this 5 ml. But in some reason, couldn't see any pellet and losing a lot of cells in supernatant. Follwoing my yield of DNA is speechlessly small.
Can anyone provide some way to get over this situation?
how could i get a cell pellet without losing
with regards
Min
This may sound very silly but I wonder if you jave tried transferring the the 5ml into 5 eppendorf tubes and spinning at 6000 rpm for 5 minutes and the removing 950ul of the supernatant from each of the spun tubes. Then resuspend the invisible pellet of cells in the remining 50ul in each tube and pool them (total about 250ul) and if you want to culture the cells, put them in a small well (24 or 48 well) with its appropriate media. If you want to get DNA then add 250ul of 2X DNA lysisbuffer to the pooled resuspended cells and follow through with the normal DNA extraction procedure.
Hope this is relevant.
Ken
So you have you tried using a centrifuge? Perhaps slightly longer and faster protocol to ensure it all pellets?
From my experience, if you have less than 100,000 cells in a 1.5 mL tube you will not be able to easily visualize the cell pellet. So in a 5 mL tube this would roughly mean you will need to have >300,000 cells.
How many cells do you use for your flow analysis? I have only used machines with 10,000 cells in analysis which you will not be able to visualize.
How much DNA yield are you getting?
What are you doing to extract DNA?
You can sort directly into something like ATL and then move from there to gDNA extraction. Instead of pelleting, which often yields a big cell loss, if you're using something like a QIAGEN kit, you can mess with the protocol and do it additively/relative to the sample volume and then vacuum through the columns instead of spinning them. I can send you a protocol for doing it this way if you want
You can use a cell filter and lyse you cell directly on a filter:
https://www.pluriselect.com/product-details/product/pluristrainer-6um.html
First of all, thank you guys all for great comments
Hi Ken,
Is it okay to spin down those sorted cells at 6000rpm?
And I also agree with using 2X or more concentrated DNA lysisbuffer
Thank for comments!
Best
Min
Hi Can,
In my case sorted cell counts were under 300,000 cells.
So as you mentioned, collecting 100,000 cells in 1.5mL volume is possible?
I guess you only collect them using sorting buffer (such as sheath, BD) ?
Also, in some case 2M cells are not vissible....
Best
Min
Hi, Robert
yes i want some protocol that i can try in different way rather than spinning down.
Could you send me ? I guess Anton attached some info below..
Best
Min
I was sorting a very rare population (0.01% of PBMCs) in 1.5ml microtubes containing 500 microleters of pure Heat inactivated FBS. and after high speed (10000g) centrifugation of 10 min I would proceed to RNA extraction and had good results. You can also sort directly in Lysis buffer.
You're very welcome! You probably know that already but I forgot to mention that sorting at minimal pressure is important for the purity of a rare population and for cell viability in general.
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