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