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I have looked in the literature (a little) and have some up with what I hope
are the appropriate reagents. I wanted to confirm these with you who have
more experience than I, and see if I am in the ball park before I go wasting
a great deal of the universities money....
Propidium Iodide (100mg) SIGMA (P4170)
RNase A ( 50mg) SIGMA (R9009)
Sodium Citrate (500g ) SIGMA (S4641)
CAM 5.2 ( 50 Test) Beckton Dickinson (349205)
anti-cytokeratin MAb
GAM-FITC (100 test) Coulter (6602159)
MsIgG2a (100 test) Coulter (6603001)
My lab tech immunology is rusty at best. Do I have the correct anitbodies
and controls?
As far as steps involved, this is what I have...
Obtain cell suspension.
Wash PBS.
Centrifuge and remove supernatant.
Fix in 67% EtOH.
Incubate with 50 microliters of CAM 5.2 for 20 minutes.
Wash with PBS
Centrifuge and remove supernatant.
Incubate with GAM-FITC at 4 degress C for 30 minutes.
Wash with PBS.
Centrifuge and remove supernantant.
Add 1-2 mls of PI/RNase/Sodium Citrate solution (I have a recipe for that
which I already use) and incubate for at least 45 minutes.
Analyze.
Similar setup for Control except using MsIGg2a instead of CAM 5.2?
Am I missing anything?
If anyone has an easier method or can correct my mistakes, I would very much
appreciate any suggestions.
Thank you.
********************
* Bill Justice *
* (billj@sky.net) *
********************
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
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