Hello,
I want to thank all those that replied...Julie you need to buy a couple of
cases of beer.
Below is a consensus summary.
Again, thanks.
-Bill
General Comments:
If you don't crosslink with paraformaldehyde, the GFP may leak out of
the cells. Unmodified GFP definitely does. If you use more than 0.5%
paraformaldehyde, the DNA curves look lousy. Even 0.5% hurts the
curves, but not too badly.
If the GFP is a fusion protein, you may be able to skip the
paraformaldehyde, but you have to check that with an experiment.
Use the correct form of GFP (farnesylated), so that it can withstand the
standard ethanol fixation for best cell cycle analysis.
Protocols:
1) Prepare single cell suspension
Fix in 0.5% paraformaldehyde in PBS 20 minutes on ice
Wash 2 times in ice cold PBS
Resuspend the pellet in the residual PBS
Permeabilize (if that's a word) by suspension of the washed pellet in -20C
90% methanol/
10% PBS (more than one ml) with vortexing
Incubate 20 minutes on ice (probably not required)
Wash 2 times in PBS with 0.1% triton X-100 and 0.5% BSA ("PBT")
Suspend cells in PBT with 100 ug/ml RNase A and PI
Hold overnight at 4C or can use 1 hour at 37C.
2) Use 1% formaldehyde (you can use paraformaldehyde, I'm sure you've
followed the debates; our formaldehyde is a 16% solution from
PolySciences) in PBS for 1 hr at 4oC, followed by a wash in PBS, then
PBS/5%FBS/0.2%Tween-20 for ~1hr, then resuspend in the same solution
containing PI (we actually use DAPI). CVs are generally in the range
of 3%.
3) DNA STAINING WITH PROPIDIUM IODIDE
Prepare in advance:
A. RNAse A solution- 2mg/ml RNAse A in PBS (pH 7.2) boil and aliquot
500ul into st. eppendorf tubes and freeze.
B. Propodium Iodide-0.1mg/ml PI in PBS (pH 7.2) with 0.6%NP40
1. Prepare cell suspensions of freshly isolated cells, or cell lines in
PBS with 3% FCS.
2. Adjust concentration to 2 x 106 cells/ml, transfer 1ml (2 x 106
cells) and centrifuge at 1100 RPM for 5 minutes.
3. Fix the cells by first resuspending the pellet in 50ul PBS+3% FCS
serum and then adding 1ml cold 80% ethanol. Resuspend well. Fix at 4C for
30 minutes. Can store at 4C for several days before staining. Pellet cells
before continuing.
4. Aspirate supernatant and resuspend the pellet in 500ul 0.1mg/ml PI+
0.6% NP-40. Vortex to mix.
5. Add 500ul 2mg/ml RNAse A to cells. Gently vortex and incubate the
cells in the dark at room temperature for 30 minutes.
6. Vortex well to resuspend cells. Filter through 85 um Nitex mesh, to
remove clumps. Keep on ice and covered until analysis.
Reference:
Cytometry 29:286-291, 1997; "Use of a membrane-localized green fluorescent
protein allows simultaneous identification of transfected cells and cell
cycle analysis by flow cytometry"; Kalejta, et al.
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