Quantification by flow cytofluorimetry of HLA class I molecules at the surface of murine cells transformed by cloned HLA genes.
A method allowing quantitative analysis of the expression of foreign antigens at the surface of transformed cells is described. Aminoethyl-Sephadex G-25 beads labelled with different amounts of fluorescein isothiocyanate (FITC) were used to calibrate an Epics V cell sorter. The quantity of FITC molecules bound per bead was found to be a linear function of relative fluorescence intensity expressed by channel number for intermediate and high levels of fluorescence and a non-linear function for low levels. The lowest limit of detectable fluorescence was 3400 FITC molecules bound per bead. Using FITC-conjugated monoclonal antibodies (m.Ab.) the number of HLA class I molecules expressed at the surface of murine LMTK- (H-2Kk) cells transfected by cloned HLA class I genes was estimated and compared with the amount expressed on human B (Raji) and T (MOLT 4) lymphoblastoid reference cells. Murine transformed cells expressed approximately 3 times less HLA class I determinants per surface unit (micrometer 2) than Raji cells and 1.4 times less than MOLT 4 cells. Similar results were obtained by Scatchard analysis of the same populations using radiolabelled m.Ab. A detailed analysis of fluorescent cells showed that 5-20% of transformed cells expressed less than 33,000 HLA class I molecules/cell whereas most MOLT 4 cells exhibited at least 280,000 molecules/cell and the majority of Raji cells more than 700,000 molecules/cell. The expression of foreign antigen did not reduce the amount of H-2Kk molecules at the surface of transformed cells (mean of 350,000 molecules/cell).
D000818:Animals; D002051:Burkitt Lymphoma; D002460:Cell Line; D003001:Cloning, Molecular; D005434:Flow Cytometry / Q000379:methods; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006680:HLA Antigens / Q000032:analysis* / Q000235:genetics; D006801:Humans; D007739:L Cells / Q000276:immunology; D008285:Major Histocompatibility Complex; D051379:Mice; D013861:Thiocyanates; D013937:Thymidine Kinase / Q000172:deficiency
D005452:Fluoresceins; D005456:Fluorescent Dyes; D006680:HLA Antigens; D013861:Thiocyanates; D013937:Thymidine Kinase; D016650:Fluorescein-5-isothiocyanate