title: Clonal isolation of chlamydia-infected cells using flow cytometry.
abstract: This manuscript describes a new technique for the microbiological cloning of chlamydia-infected cells using a fluorescence activated cell sorter (FACS). The approach exploits chlamydial acquisition of the fluorescent, Golgi-specific, stain 6-((N-7-(-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-hexanoyl)sphingosine (C6-NBD-cer). This fluorescent lipid is delivered from the Golgi apparatus to the chlamydial inclusion membrane and then to the developmental forms within the inclusion in living, infected cells. Labeling with C6-NBD-cer results in easily identifiable chlamydial inclusions that can then be analyzed and sorted by FACS. This technique was used successfully to sort individual chlamydia-infected cells into individual wells of a culture dish and, in this experimental system, resulted in the isolation of cloned chlamydial isolates. FACS-based sorting was used to isolate clonal populations of prototype strains from Chlamydia trachomatis, C. caviae and C. suis. Recent clinical isolates were also successfully cloned using FACS. The procedure is simple and rapid, with single cloning cycles being completed 24 h post-culture of a sample. It is anticipated that FACS-based sorting of live chlamydia-infected cells will be a significant technical tool for the isolation of clonal populations of any chlamydial strain.
authors.affiliation: Department of Microbiology, College of Science, Oregon State University, Corvallis, OR 97331, USA.
authors.forename: Damir T
authors.initials: DT
authors.lastname: Alzhanov
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Suchland
authors.forename: Antony C
authors.initials: AC
authors.lastname: Bakke
authors.forename: Walter E
authors.initials: WE
authors.lastname: Stamm
authors.forename: Daniel D
authors.initials: DD
authors.lastname: Rockey
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000378:metabolism; D002460:Cell Line; D002518:Ceramides / Q000378:metabolism*; D002690:Chlamydia Infections / Q000378:metabolism / Q000382:microbiology* / Q000473:pathology; D002692:Chlamydia trachomatis / Q000254:growth & development* / Q000378:metabolism; D002999:Clone Cells; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D013194:Staining and Labeling / Q000379:methods
chemical_list: D002518:Ceramides; D005456:Fluorescent Dyes; C037974:N-(7-(4-nitrobenzo-2-oxa-1,3-diazole))-6-aminocaproyl sphingosine; D009327:4-Chloro-7-nitrobenzofurazan