Quantitative Pinocytosis by FACS...
Roederer at beadle.stanford.edu
Mon Aug 3 18:24:15 EST 1998
Back when I was a graduate student with Bob Murphy (Hi Bob), we used to do this
all the time! The easiest tracer to use is relatively high molecular weight
dextran (60 KD or more) that has been conjugated to FITC or TRITC. These are
nonspecifically taken up by cells through pinocytosis (use them at 1 mg/ml
concentrations and wash extensively!) and the amount of fluroescence relates to
the amount taken up! Note that the FITC conjugate will show an additional
negative relationship to the ongoing acidification during pinocytosis; i.e., the
fluorescence gets quenched rather significantly within minutes depending on the
cell type and temperature of incubation. I did lots of experiments using both
FITC and TRITC dextran to measure not only the amount of pinocytosis
(proportional to TRITC fluorescence) but the degree of acidification (related to
the FITC/TRITC fluorescence ratio).
Nowadays, there are probably better conjugates than TRITC to use for
dextran--something like Texas Red, Alexa, or Cy3 or Cy5. TRITC is kind of
sticky and a pain to use.
More information about the Cytometry