Fixing samples to be used in the analyzer:
- Ethanol-fixation of samples
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- Place 1 x 10^6 cells into a polypropylene tube and centrifuge at 1000 rpm for 5 min.
- Resuspend the pellet with 1 mL of PBS (1x) and centrifuge 1000 rpm for 5 min
- Remove the supernatant without disturbing the pellet and add (dropwise) 1 mL of -20 C 70% EtOH to the cell pellet while vortexing.
- Keep cells on ice for 2 hrs or at -20 C until the day of the staining (cells can be stored for several weeks at -20 C)
- On the day of staining, centrifuge the samples 1000 rpm for 5 min, remove the supernatant
- Resuspend the pellet in 1 mL of PBS (1x), centrifuge again, remove the supernatant
- Resuspend the pellet in 0.5 mL of PBS (1x) + 2 mM EDTA and proceed to the staining protocol
- DNA staining with PI
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- Add 25 uL of PI solution (250 ug/ml) to 0.5 ml of PBS (1x) + 2 mM EDTA containing 1 x 10^6 cells of less, keep the samples at least 20 min on ice and covered. Use a 5 ml Falcon BD Flow tube.
Buffers for cell sorting and analysis
1x PBS (Ca2+/Mg2+ free)
0.5 – 2 mM EDTA
1-2% FCS (only for sorting)
0.2 µm filtered, store at 4 °C
Or if pH-stability is crucial
1x PBS (Ca2+/Mg2+ free)
1 mM EDTA
25 mM HEPES pH 7.0
1% FCS (heat-inactivated) or BSA
0.2 µm filtered, store at 4 °C