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of GFP and DNA Content in Fixed CellsMaterials
- Cells to be studied expressing green
fluorescent protein (GFP). Note that the same cell type without GFP is needed
as a control.
- Phospahte
Buffered Saline (recipe link
for PBS)
- Para-formaldehyde,
(recipe link for 2% Stock)
- 70%
Ethanol
- Propidium iodide
stock solution (0.5 mg/ml in PBS)
- RNAse
- 12
X 75 mm culture tubes
- Vortex
mixer
- Ice bath
- Water
bath at 37 C
ProtocolFix
cells with formaldehyde 1.
Count cells. 2. Place approximately
2 X 106 cells into a 12 x 15 mm test tube and wash them once with PBS
by centrifugation for 5 min at 300 x g at 4°C.
3.
Remove supernatant by aspiration and add 500 µl of cold PBS to the cell pellet.
Mix gently. Add 500 µl of ice cold, buffered 2% formaldehyde solution and mix
again. Incubate at 4°C for 10 minutes to 1 hour.
Lower concentrations of formaldehyde and short incubation times preserve more fluorescence.
Permeabilize
cells with ethanol
4.
Spin cells down by centrifugation for 5 min at 300 x g at 4°C, remove supernatant
by aspiration, wash once with cold 1 X PBS, then add 1 ml of 70% ethanol at -20°C
drop-wise to the cell pellet with the tube sitting on a vortex. Incubate cell
suspension overnight at 4°C. Stain
with propidium iodide 5.
Spin cells down by centrifugation for 5 min at 300 x g at 4°C,
remove supernatant by aspiration and add 1 ml of a solution containing 40 µg/ml
of PI and 100 µg/ml of RNAse. Incubate cell suspension at 37°C in the dark
for 30 min. 6. Transfer
tubes to ice bath. If needed, filter samples through a nylon mesh (BD Cat #35-2340
or 35-2235) to remove clumps before acquisition on the flow cytometer . 
This
is an example of cells expressing varying levels of GFP (y-axis) versus DNA Content
(PI, x-axis)
References:
-
Chu, YW, Wang
R., Schmid I, Sakamoto KM. Analysis with flow cytometry of green fluorescent protein
expression in leukemic cells. Cytometry 36:333-339, 1999. -
Schmid
I. and Sakamoto KM. Analysis of DNA content and green fluorescent protein expression.
In: Current Protocols in Cytometry, Vol 1, Robinson JP, Darzynkiewicz Z,
Dean P, Orfao A, Rabinovitch P, Stewart C, Tanke H, Wheeless L, eds., John Wiley
& Sons, 2001, pp. 7.16.1-7.16.10.
<---Back
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Back to Protocols Dennis
J. Young, UCSD Cancer Center Flow Cytometry Core Facility
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