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Fixing samples to be used in the analyzer:

 

Ethanol-fixation of samples

 

1. Place 1 x 10^6 cells into a polypropylene tube and centrifuge at 1000 rpm for 5 min.
2. Resuspend the pellet with 1 mL of PBS (1x) and centrifuge 1000 rpm for 5 min
3. Remove the supernatant without disturbing the pellet and add (dropwise) 1 mL of -20 C 70% EtOH to the cell pellet while vortexing.
4. 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)
5. On the day of staining, centrifuge the samples 1000 rpm for 5 min, remove the supernatant
6. Resuspend the pellet in 1 mL of PBS (1x), centrifuge again, remove the supernatant
7. Resuspend the pellet in 0.5 mL of PBS (1x) + 2 mM EDTA  and proceed to the staining protocol

 

DNA staining with PI

 

1. 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

 

Cell surface antibody staining – BD Stain Buffer-Source: cat# 554656

Intracellular antibody staining – BD Pharmingen: cat# 562574