#4 FIXATION OF EUKARYOTIC CELLS IN ETHANOL FOR CELL
CYCLE ANALYSIS BY
FLOW CYTOMETRY (FACS)
Day of Fixation of Cells
For each sample, label a separate 15ml conical tube.
Make sure you have –20C cold 70%Ethanol (keep in freezer)
1. Remove media place in a 15ml
Conical
2. Wash with 1-2ml 1x PBS place in
the same 15ml conical
3. Trypsinize the cells (15min 37C or until
lifted), place cells into
the same 15ml conical
4. Spin cells down 3min 1250 rpm
5. Aspirate media off carefully without
disturbing pellet of cells
6. Add 500ul 1x PBS and ressuspend
carefully ~4 times up and down
using p1000 pipettor
7. Turn a vortex on "medium' setting
(~4)
8. Obtain 4.5ml 70%EtOH ice-cold (from –20C
freezer TC rm)
9. Using a 5ml pipette, drop the 4.5ml
70%EtOH on top of the cell
pellet while vortexing ("adding dropwise" is the name of this
method)
10. Repeat for all cell tubes, then place all of
them in a –20C freezer
11. The cells should be "fixed" in this ethanol
solution at least 24h
prior to analysis by FACS
The day of FACS analysis:
1. Make sure to schedule an appointment
with Marilyn ahead of time.
2. Start preparing cells 1 hour ahead of
the appointment
3. Take Cells/ethanol out of –20C,
Spin 5min 1250 rpm
4. Carefully aspirate media off the top,
leaving pellet
5. Add 5ml PBS, mix
6. Spin again 5min 1250 rpm
7. Carefully aspirate media off the top,
leaving pellet
8. Now add to the pellet 500ul of Complete
PI+RNAse+Cell Cycle Buffer
for analysis, ressuspend pellet
9. Carefully push cell mix through the FACS
blue cap mesh tube to break clumps
10. Bring the tubes to FACS in foil, toom temp
ok.
A. Prepare ahead of time :
4C Cell Cycle Buffer
Make 250ml: (4C storage)
Sodium citrate
250mg
Triton X100
750ul
nuclease free water
200ml
B. Add fresh before use:
COMPLETE CELL CYCLE BUFFER (PI+RNAse) (lyses your cells and
releases
DNA PI binds and fluoresces)
For each mL of Cell cycle buffer you need, now add:
--Propidium Iodide (2mg/ml solution)
500ul
--RNAse A DNAse free (33ug/ul Sigma)
0.6ul
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