The use of fluorescence quenching in flow cytofluorometry to measure the attachment and ingestion phases in phagocytosis in peripheral blood without prior cell separation.
Flow cytofluorimetry identifies and quantifies cell markers of different leukocyte subpopulations by combining cytofluorimetry with the differences in the light scattering properties of the leukocytes in mixed populations. In the phagocytic assay, reported in this paper, the experimental conditions were selected in such a way that it was possible to analyse the phagocytic function of granulocytes in peripheral blood without time-consuming cell separation. The percentage of phagocytosing granulocytes was not dependent on the concentration of granulocytes at the selected incubation time and particle (yeast-C3b) concentration. Furthermore, it was possible to adapt a previously described fluorescence quenching technique (FQ method) to differentiate between attachment and ingestion. Crystal violet, originally used in the FQ method, could not be used in this assay due to its lysomotropic effect. Trypan blue at a concentration of 0.25 mg/ml or higher at pH 4.5 showed a plateau effect in fluorescence quenching indicating an effect on attached but not ingested particles. This assay offers a simple technique to screen the functional properties of phagocytic cells in peripheral blood.
D002462:Cell Membrane / Q000378:metabolism; D002469:Cell Separation; D003179:Complement C3b / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins / Q000494:pharmacology; D005453:Fluorescence; D006098:Granulocytes / Q000166:cytology / Q000378:metabolism; D007958:Leukocyte Count; D007962:Leukocytes / Q000166:cytology* / Q000378:metabolism; D010587:Phagocytosis; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013861:Thiocyanates / Q000494:pharmacology; D013997:Time Factors
D005452:Fluoresceins; D013861:Thiocyanates; D003179:Complement C3b; D016650:Fluorescein-5-isothiocyanate