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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