I am trying to do an ATACseq library prep on human lymphocytes and keep losing the majority of cell nuclei in aspiration steps. It was suggested I could try using Concanavalin A beads to help pellet & better visualize the pelleted nuclei, but I'm not finding protocols specifically addressing this. Anyone have any ideas?
I would not use concanavalin A for such purpose. Theoretically this procedure should not work at all.
Nuclei from lympocytes are easy to get. Is it a cell line? If lymphocyes from blood, make sure they are "fresh"
If you use a cell line or fresh lmphocytes and proper lysis buffer, nuclei are easily isolated (and visible after centrifugation to avoid aspirating them). I also wonder how you perform aspiration. Mild! or manually using a pipette with thin tip end.
Thank-you for your reply Rafael. That was my impression after reading up on uses & protocols for concavalin A, however a post-doc suggested it, so thought I'd ask the community.
I have been modifying my spin conditions & aspiration steps, and think I'm finally onto a protocol that seems to be working with less than 5% loss.
For anyone interested, I doubled the centrifuge speed (from recommended 500xg to 1000xg), doubled the spin time (from recommended 5 min to 10 min), both of which seem to the cells pellet more visibly. I then incorporate multiple pipetting steps to aspirate, with successively smaller tips, each time leaving a small volume behind with the pellet. I save all my supernatants and count cells in each. The majority contain 1-2% or less of my starting cell #'s, making me more confident that this is working.
Dear Selene, another solution to avoid cell loss during washing steps has recently been introduced by Silicon Biosystems, it's called VRNxT. it's not based on aspiration but on split centrifugal force, an innovative solution. You can find some more info on the web page http://www.siliconbiosystems.com/vrnxt . It only works on 200uL PCR tubes (no 1.5mL eppendorf tubes) but it can remove 99% of supernatant from a pellet of even a single cell without loosing any cell. Let me know if you could be interested in a demo at your laboratory. best regards, Raimo.
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