We simply stain the lysed and washed whole blood leukocytes with an FITC goat anti-human IgG and IgM. We include a normal control individual at the same time to eliminate most non-specific staining and gate on the neutrophils. Markers are set on the normal neutrophil FL1 fluorescence so that <5% of the neutrophils are positive. This percentage is subtracted from the corresponding percentage for the patient. >10% staining for the patient is considered positive. You could also do FRET staining to completely eliminate Fc receptor binding, but this is fairly complex. See Koksch et al. J. Immunol. Methods. 187: 53-67, 1995, for a comparable FRET method applied to platelets. This should be easily adapted to neutrophils. If you have more questions, e-mail me. Best regards, Tony Bakke Antony Bakke, PhD Oregon Health Sci Univ Dept of Pathology bakkea@ohsu.edu >>> "Newsom, Brian S." <BSNEWSOM@txccc.org> 04/15 7:19 AM >>> I have an investigator that wants to look at anti-neutrophil antibodies on circulating neutrophils. Does anyone have an antibody combination or technique for doing this? Any advice is appreciated. Brian Newsom Director, Flow Cytometry Center for Cell and Gene Therapy Baylor College of Medicine
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