I am currently sorting neuronal (NeuN+) and non-neuronal (NeuN-) nuclei from human post-mortem brain tissue using a FACS Aria II. The FACS data look as expected: we gate for positivity for the nuclear stain PI as well as a positive + negative gate for NeuN.
I typically sort tubes containing 300-800k nuclei, which are kept on ice at all times. The nuclei are suspended in PBS (FACS sheath fluid). However, if I try to spin down the nuclei (10 min at 450g, swinging bucket rotor), I don't see any pellets (same thing for longer duration or higher speed). I assumed that the pellets were too small to be visible, but if I spin down and continue with DNA/RNA purification, I obtain an amount of nucleic acids that is far smaller than expected based on cell number. If I reflow the sorted sample right after FACS, it looks fine. Apparently the nuclei get lost after sorting...
My guess is that the nuclei either remain stuck to the tube walls, or somehow lysate, maybe due to inadequate buffering conditions.
Could you recommend 1. a method to stop the nuclei from sticking (e.g. specific types of tubes, treatment with BSA) or 2. buffer conditions for sorted nuclei (e.g. adding sucrose, BSA or salts to the sorted nuclear suspension)?
Hi Manfredi,
Its always tough to see nuclei pellet even though the sorted population number is higher. They are in various sizes and can be quite small. Nothing to worry. What I would suggest is sort the nuclei basedbon DAPI or PI positive fraction in facs and then centrifuge for 10 min at 1000rpm. Slowly remove the supernatant and leave about 50ul of volume behind. Mix and take 10ul, add 10ul trypan blue and see how many nulei you can see under microscope. This will give you the recovery rate after sorting.
As far as sorting is concerned, it is recommeded that either use low binding tubes or coat your tubes with any buffer i.e., before sorting top up tubes to maximum with buffer. Remove buffer right before sorting. This will minimize nuclei lossbon sides. Make sure you mix and short spin after sort.
Don't sort in lysis buffer, that would kill them all. Sort in 2% fbs. It all depends on what you want further downstream.
Good luck
Regards,
Naush
Hi,
Do you count the nuclei before and after the sorting?
Can you partage your protocol?
Many thanks
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