When quantifying phagocytosis, rather than quenching extracellularly bound signal, we use a control tube that is incubated on ice. Any fluorescence in these cells should be due to surface bound particles (whether it is e. coli, dextran, or ova), and this can be compared to the 37 degree sample. kb -- Keith Bahjat Graduate Assistant University of Florida College of Medicine Gainesville, Florida Voice: (352) 392-4887 Fax: (352) 392-5393 e-mail: kbahjat@ufl.edu > From: "John Waitumbi" <waitumbi@net2000ke.com> > Organization: Walter Reed Project > Reply-To: "John Waitumbi" <waitumbi@net2000ke.com> > Date: Thu, 13 Apr 2000 17:39:14 -0700 > To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu> > Subject: Re: Quantification of macrophage phagocytosis > > > Raymond > > Try quenching FITC-fluorescence of extracellular bacteria by addition of > trypan blue or crystal violet (trypan blue 1 mg/ml or crystal violet 0.8 > mg/ml at pH = 7.4). Blut (1984) 49: 315-323. Remember to flush the machine > with bleach. > > John > > ------------------------------------------------------------------------ > Dr. John N. Waitumbi > Walter Reed Project > P.O. Box 30137 Nairobi > Kenya > Phone +254 35 22942 > Email:waitumbi@net2000ke.com > Fax +254 35 22903 > Electronic Fax Service: 1-603- 947-4913 > ----- Original Message ----- > From: D Raymond <dpr4w@virginia.edu> > To: cyto-inbox > Sent: Tuesday, April 11, 2000 7:33 AM > Subject: Quantification of macrophage phagocytosis > > >> >> I am trying to quantify macrophage bacterial phagocytosis but have had > numerous problems. >> I am trying to quantify the phagocytosis of E. coli by the murine > macrophage cell line >> RAW 264.7. I have tried using FITC labelled E. coli but have had problems > quenching >> external bacteria. The RAW cells take up the quenching agent (ethidium > bromide) almost >> immediately They do appear viable when I quantify them by tryphan blue > exclusion and, >> under similar conditions, we see significant cytokine production. If > anyone has any >> experience with this technique I would appreciate any input. Thank you > for your time. >> -Daniel Raymond (Charlottesville, VA) >> >
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