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In response to your query about flow cytometric analysis of phagocytosis:
I have developed a flow method for the analysis of phagocytosis of FITC
labelled E. coli by peripheral blood neutrophils. I haven't tried it with
macrophages but it works really well with neutrophils. The results have
not yet been published (manuscript is still in preparation).
Basically, FITC labelled bacteria are incubated in duplicate with the
neutrophils. One of the duplicates contains cytochalasin D, which prevents
phagocytosis. Fluorescence from FCM analysis of this tube indicates the
extent of adherence of bacteria to the outside of the neutrophils.
Phagocytosis is allowed to proceed in the second tube. Fluorescence from
FCM analysis of this tube indicates the extent of phagocytosis PLUS
adherence of bacteria. The difference between the two gives an indirect
assessment of the amount of phagoctosis. The advantage of this yechnique
is that you do not have to mess around with quenching agents etc. to
destroy the fluorescence due to adherent bacteria.
Another method is that published by Cathy White-Owen et al. (1992) Rapid
whole blood microassay using flow cytometry for measuring neutrophil
phagocytosis. J. Clin. Microbiol. 30:2071. I had trouble with the method
myself, but it might work in your hands.
It is difficult in a short e-mail to get all the details over clearly.
However, if the info in this message sounds like the kind of stuff you are
after, contact me by phone, fax or e-mail and we can discuss things in more
detail.
See ya!
BTW, I originally sent this message directly to yopur e-mail address but
the message bounced back as being undeliverable. Is the address on your
original message to the list correct?
Regards,
Dianne Budd
Room 308:206 Tel: (09) 351 7177 Fax: (09) 351 2342
Int: 619 351 7177 619 351 2342
School of Biomedical Sciences
Curtin University of Technology
GPO Box U1987 Perth 6001 WA
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