Quenching of intracellular autofluorescence in alveolar macrophages permits analysis of fluorochrome labelled surface antigens by flow cytofluorometry.
We report a new technique in which the autofluorescence of alveolar macrophages from smokers is quenched by crystal violet. This technique permits immunostaining of surface antigens of these cells and enables the stained cells to be analysed by flow cytofluorometry. The variable solubility of crystal violet makes it important to characterize the crystal violet solution by its quenching properties and not rely on the assumed concentration of dissolved dye. High concentrations of crystal violet lowered the number of cells and gave a decreased amount of surface antigen (CD11b). However, a lower concentration of crystal violet could be used if the cells were fixed with paraformaldehyde (4%) and the membranes were permeabilized with n-octyl-beta-D-glucopyranoside (0.6%). Using a phagocytic model with FITC-conjugated particles we were able to show that this treatment gave an efficient permeabilization of phagolysosomal membranes.
Halldén
G
G
Department of Clinical Immunology, Karolinska Hospital, Stockholm, Sweden.
Sköld
C M
CM
Eklund
A
A
Forslid
J
J
Hed
J
J
D000954:Antigens, Surface / Q000032:analysis*; D002463:Cell Membrane Permeability / Q000187:drug effects; D003902:Detergents; D005434:Flow Cytometry / Q000379:methods*; D005840:Gentian Violet / Q000494:pharmacology; D005960:Glucosides / Q000494:pharmacology; D006684:HLA-DR Antigens / Q000096:biosynthesis; D006801:Humans; D016177:Macrophage-1 Antigen / Q000096:biosynthesis; D016676:Macrophages, Alveolar / Q000276:immunology*; D010588:Phagosomes / Q000187:drug effects; D012907:Smoking; D014343:Trypan Blue / Q000494:pharmacology
D000954:Antigens, Surface; D003902:Detergents; D005960:Glucosides; D006684:HLA-DR Antigens; D016177:Macrophage-1 Antigen; C018619:octyl-beta-D-glucoside; D014343:Trypan Blue; D005840:Gentian Violet