Phagocytosis by flow
Tom Mchugh (mchugh@pangloss.ucsf.EDU)
Wed, 26 Jun 1996 18:09:20 -0700 (PDT)
For phagocytosis by flow we have done this on-and-off for many years. We
generally use FITC labeled bacteria or yeast with trypan blue (final
0.05%) to quench surface, non-ingested organisms. The important steps
are to have organisms at the right concentration, we use 10-20 organisms
per cell (phagocyte). The FITC labeling step affects how well the
organisms are ingested (heat, formlin, grown with FITC, etc.). The
incubation of cells with organisms varies but we generally use 15
minutes at 37C with constant mixing. A wash with cold PBS stops the
reaction to allow for centrifugation and washing. The trypan blue seems
to work well and people have used PI as well (surface organisms
fluoresce red, intact live cells do not fluoresce). If using PI it is
good to make sure your FITC organisms do not fluoresce too much in the
red region. The trypan blue reference we used was Sahlin, et al. J.
Immunology Meth 60:115-124, 1983. A good recent paper on phagocytosis is
White-Owen J. Clin. Micro 30:2071-2076, 1992. Many other papers notably
by Bassoe. Serum/antibody can be used prior to incubation with cells for
some attempt at measuring opsonization.
We get FITC labeled organisms (bacteria and yeast) from Pinnacle
Biosystems (415) 548-0490 and they seem to be working well.
Tom McHugh
Dept. Lab. Medicine
University of California, San Francisco
mchugh@labmed.ucsf.edu
CD ROM Vol 2 was produced by staff at the Purdue University Cytometry Laboratories
and distributed free of charge as an educational service to the cytometry community.
If you have any comments please direct them to Dr. J. Paul Robinson, Professor & Director, PUCL,
Purdue University, West Lafayette, IN 47907. Phone:(317) 494-0757; FAX (317) 494-0517; Web http://www.cyto.purdue.edu
EMAIL robinson@flowcyt.cyto.purdue.edu
