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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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