Fellow flowers H-E-L-P!!!,
Does anyone have experience with flow cytometric analysis of fresh liver
tissue? I am attempting to dual stain using an indirect method (goat
anti-mouse fitc) followed by propidium iodide/RNAse.
I am mashing the liver (in RPMI media) using a Tekmar-Dohrmann Stomacher,
followed by lysis with 1 X ammonium chloride lysing buffer.
To fix and permeabilize, I am using 1% paraformaldehyde/20 fg/ml
lysophosphatidyl choline at RTx for 2 min., followed by absolute methanol for
10 min. on ice, followed by .1% NP-40 for 5 min. on ice.
The cells are then stained using 200 fl Santa Cruz monoclonal antibodies at a
1:20 dilution incubated on ice for 15 minutes. The cells are then washed in
PBS/.1% azide followed by incubation with 200 fl goat anti-mouse Fitc at a
1:40 dilution for 15 minutes on ice in the dark.
The cells are washed as above, and incubated with a solution containing 5
fg/ml propidium iodide/50 fg/ml RNAse for 15 min. on ice in the dark.
I have been analyzing the cells on a BD Facsan equipped with Cellquest
software.
So far, I have run into several problems:
1) Very poor viability after lysing. Using trypan blue as a viability stain
while counting on a hemacytometer, the liver cells appeared to be all
dead. There are a fair amount of red cells in the liver homogenate,
but I'm wondering if it would be prudent to skip the lysing step, and
threshold out the red cells during analysis?
2) High background fluorescence. I have tried to block by diluting the
secondary antibody in both 10% and 20% heat inactivated goat serum, and
also by incubating the cells with 30 fg/ml goat Ig for approximately 15
min. on ice prior to adding the secondary antibody. This is my first
experience working with murine cells. Do they innately exhibit a high
degree of autofluorescence?
3) Compensation. I am experiencing diffuculty compensating Fitc and PI.
There seems to be a high degree of overlap. Any suggestions?
Also, if anyone has any experience with a similar procedure using frozen liver
tissue, that would be great.
Thanks in advance for your input,
Connie Wagner
Monsanto Environmental Health Lab
645 S. Newstead Ave.
St. Louis, MO 63110
Email: cawagn@ccmail.monsanto.com
Phone: (314) 694-7971
Fax: (314) 694-7938
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