[Histonet] Re: cell pellet preparation

[Galina Deyneko]

I attach my protocol for cell pellet 

 
Preparation of formalin fixed Cell pellets.
1. Grow up approximately 5-1o millions cells.
2. Centrifuge the cells with the medium in conical tubes for 10 minutes with speed 1000rpm.
3. Remove supernatant, wash once with 1X PBS at RT, centrifuge as before and discard PBS supernatant.
4. Place 2 ml or more of 10 % formalin in tube, flick to break up pellet, and re-suspend the cells in formalin and fixed 20 minutes at RT.
5. Centrifuge as before and remove formalin supernatant.
6. Wash once with 1X PBS, at RT, centrifuge as before and discard PBS supernatant.
7. Add 1 ml of 1X PBS, carefully re-suspend the cells and transfer to 1.5 ml Eppendorf tube.
8. Microfuge 10 minutes at 0.8 xg (2900 rpm) and remove as much PBS supernatant as possible.
9. Warm the Histogel (Allan Scientific # HG4000-012) in microwave oven 10-20 seconds till Histogel turns to liquid. Let it cool a bit.
10. Re-suspend cells in 100-150 µl of warm Histogel, leave pellet undisturbed for at least 20 minutes in wet ice for gel solidifying.
11. Remove the solid gel pyramid with sharp wood stick, wrap in histo paper place in cassette and process 
 
 
Galina Deyneko
Novartis, Cambridge, MA
 
617-871-7613 w