Dear Flowists, We are currently working with alveolar macrophages, which express a huge amount of autofluorescence (nice diagonal background on Fl-1 v Fl-2). Upon staining eg. with a CD14PE or CD11cPE it becomes almost impossible to qualify the stain, due to this high background. My question is: Is there any possible way to quench this fluorescence in alveolar macs (is the background carbon particles phagocytosed in the alveoli by the macs...maybe, but how can I reduce it...is it possible to quench intracellular particles, pre stain) I have attempted to use compensation, with reduction of PMT volts, but I have had as much luck as a fish out of water breathing. Is this background going to be as high if I stain with non PE or FITC (does the background bleed into higher wavelengths eg texas red or rhodamine ~630 nm). Saturation with antibody has very little effect in our case. So many questions.... I would love to hear from other frustrated macists and their reslove to this problem (if there is one). Fanx for your time Geza Paukovics - Flow Laboratory - AIDS Pathogenesis Research Unit Macfarlane Burnet Centre for Medical Research .***. .***. .** PO Box 254 _--_|\ * | | | * * | | Fairfield, VIC 3078 / \ * * | | | * * | | | AUSTRALIA. \_.--._/ * * | | | * | | | * ph. (+61 3) 9282 2132, O '***' '***' FAX (+61 3) 9282 2100
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