title: A novel flow cytometry-based tool for determining the efficiency of human cytomegalovirus infection in THP-1 derived macrophages.
abstract: Human cytomegalovirus (hCMV) is a ubiquitous pathogen that causes congenital infection and severe infections in immunocompromised patients. Chronic hCMV infection may also play an important role in immunosenescence and adverse health outcomes in older adults. THP-1, a human monocytic cell line and its derived macrophages serve as a useful cell culture model for mechanistic studies of hCMV infection and its underlying biology. A major methodological challenge is the lack of a quick and reliable tool to accurately determine the efficiency of hCMV infection in THP-1 derived macrophages. In this study, we developed a flow cytometry based method using commercially available monoclonal antibody (MAb) against hCMV immediate early (IE) antigen that can accurately determine infection efficiency. We used 0.5% formaldehyde for fixation, 90% methanol for permeabilization, and incubation with FITC conjugated MAb at 37°C. The method was tested by hCMV infection with laboratory Towne strain in the presence or absence of hydrocortisone. It was also compared with the routine flow cytometry protocol using Cytofix/Cytoperm solution and with immunofluorescence. The results indicate that this new method is reliable and time saving for accurate determination of infection efficiency. It may facilitate further investigations into the underlying biological mechanisms of hCMV infection.
authors.affiliation: Division of Geriatric Medicine and Gerontology, Johns Hopkins University School of Medicine, 5505 Hopkins Bayview Circle, Baltimore, MD 21224, USA.
authors.forename: Huifen
authors.initials: H
authors.lastname: Li
authors.affiliation: Zhejiang Provincial Key Lab of Geriatrics, Zhejiang Hospital, Hangzhou, Zhejiang Province, China.
authors.forename: Genxiang
authors.initials: G
authors.lastname: Mao
authors.affiliation: Division of Geriatric Medicine and Gerontology, Johns Hopkins University School of Medicine, 5505 Hopkins Bayview Circle, Baltimore, MD 21224, USA.
authors.forename: Joshua
authors.initials: J
authors.lastname: Carlson
authors.affiliation: Division of Geriatric Medicine and Gerontology, Johns Hopkins University School of Medicine, 5505 Hopkins Bayview Circle, Baltimore, MD 21224, USA. Electronic address: sleng1@jhmi.edu.
authors.forename: Sean X
authors.initials: SX
authors.lastname: Leng
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology; D000956:Antigens, Viral / Q000032:analysis*; D002460:Cell Line; D003587:Cytomegalovirus / Q000254:growth & development*; D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique; D006801:Humans; D008264:Macrophages / Q000821:virology*; D014773:Virology / Q000379:methods*
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral
keywords: Flow cytometry; Infection efficiency; THP-1; hCMV