Fluorescent protein fixation for FACS

Fixation Technique

!!! For this protocol, work in the hood !!!

Based on this protocol

1. Spin cells in eppendorf (5000xg) or plate (2250xg)
2. Remove supernatant
3. Add 100 µl of paraformaldehyde and vortex
4. Incubate at RT for 15 minutes
5. Spin cells and remove supernatant.
6. Wash once in 300µl KPO4/sorbitol (1.2M for iso-osmolarity with fission yeast).
7. resuspend in ~50-100µl KPO4/sorbitol.
8. Store cells in refrigerator for up to one month.
9. Before use, vortex well.

4% formaldehyde dilution

1. Take the formaldehyde stock solution (20%) in the chemical room from the fume cabinet. NOTE: paraformaldehyde is bad for you, so work in the fume hood!!
2. Add 10ml stock solution with 40ml ddH₂O in a falcon tube
3. Mix by inversion
4. Filter sterilize and store at 4 degrees

1M KH₂PO₄

68 g per 500 ml water

Autoclave

1M K₂HPO₄

87 g per 500 ml water

Autoclave

Potassium phosphate

83.4 ml K₂HPO₄ 1M

16.6 ml KH₂PO₄ 1M

2M sorbitol

Dissolve 182 g sorbitol per 500 ml water (heat water)

Autoclave

KPO₄/sorbitol

60 ml 2M sorbitol (final concentration 1.2M)

10 ml 1M potassium phosphate

30 ml water