Skip to main content
Figure 4 | Virology Journal

Figure 4

From: Three-dimensional Huh7 cell culture system for the study of Hepatitis C virus infection

Figure 4

Robust HCVcc infection in 3-D Huh7 RWV cultures. (A) Huh7 3-D aggregates were infected with HCVcc JFH-1 at an MOI of 0.01 FFU/cell 1 day post seeding in the RWV. Culture supernatant and intracellular RNA were collected at the indicated times p.i. Normalized intracellular HCV RNA copy numbers, displayed as HCV RNA copies/μg total cellular RNA (line), were determined by RTqPCR. Infectivity titers, expressed as FFU/ml (bars), were determined by immunohistochemical analysis of 10-fold serially diluted culture supernatants on naïve Huh7 cells. (B) Indirect immunofluorescence analysis of HCV E2 expression in HCV-infected 3-D Huh7 aggregates 14 days p.i. Additional 3-D Huh7 cultures were infected on day 1 (C), 7 (D) or 14 (E) post seeding in the RWV. Aggregates were fixed 10 days p.i. and stained with a human anti-E2 antibody (C1) and anti-human-Alexa 555 secondary antibody. Images were captured via confocal microscopy (630×, Zeiss LSM 510, Germany) and Zeiss LSM Alpha Imager Browser v4.0 software (Zeiss). Image brightness and contrast were adjusted using Adobe®Photoshop® (San Jose, CA). (*) = 100 μm microcarrier bead. Small vertical panels represent x-z sections of larger x-y sections, which were compiled by taking 0.5 μm steps through corresponding x-y sections. Red lines indicate the plane from which the z section was taken. The scale bar equals 20 μm.

Back to article page