Flow Cytometry Facility

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 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

Cancellation Policy:  A booked session on the analyzer LSRFortessa X-20 can be cancelled free of charge until 12 hours before the actual starting time. A booked session on the cell sorter FACSAria Fusion can be cancelled free of charge until 24 hours before the actual starting time. Otherwise, the normal usage fees will apply. If a user does not use the instrument through the entire booked session, usage fees will be calculated according to the booked time.