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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