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YOU COULD TRY GOING DOWN AND DIRTY ----
If the tumors are visible to the naked eye or can be seen under a dissecting
scope you could try doing a needle biopsy of them by using a 25-27 guage
needle on a 1 ml tuberculin syring with a about .2 ml of tissue culture media
or phosphate buffered saline with a little fetal calf serum in it.
Just sticking it in and out of the tumor with slight suction several times.
You should get enough cells for flow this way. Be sure to do some negative
controls the same way on untreated mice so you can recognise the small
populations you may be looking for from background. You may be able to use
the surrounding tissue as a control if the tumors are well defined and
isolated, but untreated tissue should be run as well.
You may need to lyse the red cells in your suspensions with a red cell lysing
solution like Sigma Chemical Co. Cat # R7757. If endodoxin stimulation
doesn't matter you can make your own. It's 8.3 grams/liter of ammonium
chloride in 0.01 Molar Tris-HCL buffer at about 7.2 ph I think. Since I do
lymph nodes and spleens for white cells, endotoxin stimulation is the last
thing I want, so I buy the commercial stuff. It's cheap. A 100 ml bottles
cost $8.80 0.1 for about 1-2 minutes, then a couple of quick washes sould
give you a nice cell suspension without red cells. You want to get rid of
the red cells so the autoflourense from hemoglobin doesn't interfere with your
floursent antibodies. You may not need to do this if your livers aren't
particularly bloody. If you don't need the blood in the liver, exsaguinate
the mice first, then take the liver out. You might not have to worry about
the red cells that remain. I'd put the livers in some tissue culture media
until your ready to process them.
You might want to block the cells with a product called FcBLOCK from
Pharmingen if you are using a directly labelled monoclonal antibodies. It
should help reduce your nonspecific background. It blocks the FcII and FcIII
receptors or you could try 10% mouse sera for about 5 minutes before you stain
with your flourescent labelled antibodies.
FINALLY, TALK TO YOUR LOCAL FLOW CYTOMETRIST(S), HE/SHE PROBABLY HAS DONE
TISSUE LUMPS AND BUMPS FOR BOTH SURFACE AND NUCLEAR MARKERS. ONCE YOU GET
THROUGH THE EXTERIOR, MOST OF US TRY TO BE A REASONABLLY FRIENDLY, Its just
that we don't like "OSPD'S" messing up our equipment. (That's One Shot Prima
Donna's/Don's for those of you unfamiliar with the vernacular)
Lots of luck to you.
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