Concentrating low volume of cells - (Nov/17/2012 )
I'm trying to concentrate a low number of LNCaP cells (50-200) in a large excess volume (1.5mL of PBS + 1%BSA+1mM EDTA), for culture in 96-well plates. I've tried spinning down the eppendorf containing the cells (740xg for 5 min), and pipetting the 1.5mL into the well plates directly, but cell recovery is very low (1-5%). I'm sure there is a smarter way to do this and would welcome advice, the only constraint being there is no way I can bring the intial volume down from 1.5mL, it's a limitation of the equipment I'm using. Thank you.
After you pellet your cells, how do you remove the supernatant before resuspending the cells? Perhaps you are losing many of your cells when you aspirate the supernatant?
I would try gently pouring it off, trying not to disrupt the cell pellet, and if that means leaving a little behind, that's ok.
I use a 200 microliter pipetman to aspirate off about 500 microliters of fluid when I attempt to pellet the cells. I then plate the rest of the volume 200 microliters at a time in the 96-well plate. Results from that, and from just plating the entire volume with no centrifugation (1.5mL), are about the same. Is there a better way to remove supernatant? Thanks for the suggestions, the problem has been really frustrating for me.
Hmmm. Ok. What about increasing the centrifugation time to 10 min?
Also, I have found that centrifuge speeds that I use for my big tubes (15 and 50ml) in the larger benchtop centrifuges pellet my cells MUCH better than equivalent rcf values in a microfuge. I don't know why this is (350xg for both).
I get around it by increasing the speed for the microfuge- but by this stage my cells are usually fixed and so I don't have to worry about viability.
Hopefully someone else has some tips for you
I agree with leelee, I get better recovery with larger tubes and increased time. I have some crazy cells that are very difficult to pellet, and if I don't spin them for 20 minutes, I lose most of them that are still suspended in solution. For these difficult to pellet cells, when I aspirate the medium, I take off enough medium to allow me to lay the tube on it's side, then aspirate as much liquid as I can safely, keeping my tip well away from the cells leaving some medium behind.
As for cfg speed, depending on the cell line, I pellet mine at 800 - 950 rpm without losing viability. Will your cells tolerate a higher rpm/g's?
So would I put the 1.5mL directly into a 15mL conical tube to spin down, or just place the 2mL eppendorf in the larger centrifuge? We have the buckets to do both. I will try extending the time to 20min, and try higher g's. We haven't specifically looked to see what LNCaPs can handle, but that should be straightforward enough, I'll just need lots of tubes!
Do not try higher RCF (=g - but it's not gravity so this is referred to as relative centrifugal force) - this will rip your cells apart by shear forces, which is possibly why you are getting low recovery anyway! What will happen if you spin them at high RCF is that you will get the smaller cells and debris smearing up the back/top side of the tube which will make it harder to re-suspend. The maximum you want to spin cells at and have them remain fully viable is about 300 RCF.
The larger tubes thing will help some and extended pelleting time can help, but really - don't spin at higher RCF!
I'm sorry for the confusion, I use 100 - 200 x g to pellet cells with high recovery and viability. For difficult to pellet cells, increasing time works well.
Thanks for the catch Bob I re-read the original post and see that Engi was spinning cells at 750xg not RPM, my very very bad
Thanks! It's actually 450xg, hit the wrong number on my keypad and noticed too late to go back and edit.