Hello Michele I have used a protocol that uses Formaldehyde fixation for 15 min. (for the surface stain), this is washed off and cells are fixed in Ethanol overnight or longer (for a nicer PI cell cycle stain). Then the cells were permed with .2% Tween-20 and incubated with intracellular antibodies, washed and stained with PI for at least an hour or more. They look great this way, although compensation can be tricky especially for the PI, if there is too much PI or too few cells. HOWEVER, the ethanol destroys APC, so I have used FITC, PE and PI in FL3. You can use APC, if it will bind after the Fixation steps. Hope this helps. Artur ********************** Artur Plett, PhD Post-doctoral fellow IU School of Medicine Div. of Hematology/Oncology Indianapolis, IN 46202 -----Original Message----- From: Michele.Nadaf@mcmail.vanderbilt.edu [mailto:Michele.Nadaf@mcmail.vanderbilt.edu] Sent: Wednesday, May 09, 2001 1:25 PM To: cyto-inbox Subject: DNA and cell surface staining Dear Flow-ers. I have been approached by someone wanting to analyze cell cycle (using PI) and simultaneous surface stains using CD21-FITC and a biotinylated HSA on a dual laser FACSCalibur. (488 nm and 635 nm lasers) I see no problem with the FITC ab, however am seeking a recommendation for the unconjugated ab, perhaps APC? Also would there be any major diffuculties with using a biotinylated ab with this kind of stain, i.e. non specificity and what could they do to avoid these problems? I have done this before with FITC conjugated surface Ab after several tries to optimize the fixation procedure, (either loss of surface stain or insufficient PI stain) finally getting it to look nice. However, have not tried it with a 2nd Ab that was biotin. Any suggestions on protocols that have worked best for you? Any help with the previous questions would be appreciated. Thank you. Michele
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