Hi everyone,

I'm currently trying to isolate nuclei from mouse brain tissue for downstream FACS. I’d appreciate any advice or thoughts on whether my yields seem within a reasonable range.

Here are two trials I did:

• 22.1 mg brain tissue Gradient: 1.6 M – 1.8 M – 2.3 M sucrose Final suspension: 140 μl PI staining: 1.62 × 10⁵ nuclei/ml
• 38.1 mg brain tissue Gradient: 1.6 M – 1.8 M sucrose Final suspension: 180 μl PI staining: 5.23 × 10⁴ nuclei/ml (Possibly underestimated — some nuclei appeared as hollow or ring-shaped PI+ signals that might have been excluded during analysis. What causes these hollow or ring-like PI signals?)

Both runs were done using a SW25.5 rotor (which I later realized is not a swinging-bucket — I'll switch to SW41 in the next round).

My question is: ➡️ Are these yields typical for this amount of tissue? ➡️ Should I just increase the input tissue for sorting, or is this too low and something needs troubleshooting?

➡️ What causes these hollow or ring-like PI signals in 388?

Thanks so much in advance!

Best, Raven