Fixation and long-term storage of human lymphocytes for surface marker analysis by flow cytometry.
A method to preserve stained human lymphocytes for subsequent cell surface analysis by flow cytometry (FCM) is described. Cells stained with fluorescein isothiocyanate (FITC) and phycoerythrin (PE)-conjugated monoclonal antibodies and then fixed in 1% paraformaldehyde, followed by extensive washing and resuspension in 1% BSA medium, could be stored at 4 degrees C for at least 2 weeks prior to FCM analysis without significant alteration in the light scatter or fluorescence properties of the cells. Furthermore, the method was also suitable for analyzing lymphocytes that express T-cell activation markers in certain disease conditions. In addition, we have identified monoclonal antibody combinations that discriminate different lymphocyte subsets that are satisfactory for multiparameter analysis after 2 weeks of storage. This method should prove useful for enumerating lymphocyte subsets in field study sites remote from flow cytometry laboratories.
D000911:Antibodies, Monoclonal; D000943:Antigens, Differentiation / Q000032:analysis / Q000276:immunology; D000954:Antigens, Surface / Q000032:analysis*; D001793:Blood Preservation / Q000379:methods*; D005404:Fixatives; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005557:Formaldehyde; D006684:HLA-DR Antigens / Q000032:analysis; D006801:Humans; D008180:Lupus Erythematosus, Systemic / Q000097:blood; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000032:analysis*; D010799:Phycoerythrin; D011108:Polymers; D011657:Pulmonary Eosinophilia / Q000097:blood; D013861:Thiocyanates
D000911:Antibodies, Monoclonal; D000943:Antigens, Differentiation; D000954:Antigens, Surface; D005404:Fixatives; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006684:HLA-DR Antigens; D011108:Polymers; D013861:Thiocyanates; D010799:Phycoerythrin; D005557:Formaldehyde; D016650:Fluorescein-5-isothiocyanate; C003043:paraform