intra cellular flow cytometry kit  (Cell Signaling Technology Inc)

Journal: Genes to Cells

Article Title: Establishment of a Novel Caco‐2‐Based Cell Culture System for Human Sapovirus Propagation

doi: 10.1111/gtc.70007

Figure Lengend Snippet: Susceptibility of various cell lines to HuSaV infection. (A) Changes in HuSaV RNA copy numbers in the culture supernatants of HuTu80 and HEK293T cells following inoculation with a HuSaV GI.1 (AH20)‐positive stool suspension (~4 × 10 7 copies/well in 96‐well plates). Each dot represents individual data points; bars indicate the geometric mean value of the HuSaV RNA copy numbers; error bars denote the geometric standard deviation (SD). This experiment was performed once with five technical replicates. (B) Changes in HuSaV RNA copy numbers in the culture supernatants of Caco‐2, HCT15, HCT116, Caco‐2/Cas9, and C2BBe1 cells following inoculation with a HuSaV GI.1 (AH20)‐positive stool suspension (~2 × 10 6 copies/well in 96‐well plates). Each dot represents individual data points; bars indicate the geometric mean HuSaV RNA copy numbers; error bars denote the geometric SD. This experiment was performed once with five technical replicates. (C) Immunofluorescence staining of the viral protein VP1 in HuTu80, HEK293T, Caco‐2, and Caco‐2/Cas9 cells at 3 dpi with a HuSaV GI.1 (AH20)‐positive stool suspension; upper panels: 4× objective lens, lower panels: 40× objective lens. (D) Flow cytometry analysis of Caco‐2 and Caco‐2/Cas9 cells infected with a HuSaV GI.1 (AH20)‐positive stool suspension at 4 dpi. Blue: VP1‐negative cells (uninfected), red: VP1‐positive cells (infected).

Article Snippet: The cells were pelleted by centrifugation, fixed with 4% formaldehyde for 15 min, and permeabilized with methanol for 10 min at 4°C using the Intra Cellular Flow Cytometry kit (Cell Signaling Technology, MA, USA).

Techniques: Infection, Suspension, Standard Deviation, Immunofluorescence, Staining, Flow Cytometry

Journal: Genes to Cells

Article Title: Establishment of a Novel Caco‐2‐Based Cell Culture System for Human Sapovirus Propagation

doi: 10.1111/gtc.70007

Figure Lengend Snippet: Immunofluorescence and flow cytometry analysis of highly HuSaV‐susceptible Caco‐2MC cells. (A) Immunofluorescence staining at 4 dpi with HuSaV GI.1 (AH20) on cloned cells derived from Caco‐2/Cas9 cells; (left) clone M (Caco‐2M), (right) clone P (Caco‐2P); blue: Nuclei (Hoechst). (B) Flow cytometry at 4 dpi following infection with HuSaV GI.1 (AH20) in Caco‐2M, Caco‐2MC, and Caco‐2ME cells; blue: VP1‐negative cells (uninfected), red: VP1‐positive cells (infected). (C) Immunofluorescence staining of Caco‐2MC cells at 4 dpi following infection with HuSaV GI.1 (AH20). (D) Flow cytometry at 4 dpi following infection with HuSaV GI.1 (AH20) in Caco‐2PG cells.

Article Snippet: The cells were pelleted by centrifugation, fixed with 4% formaldehyde for 15 min, and permeabilized with methanol for 10 min at 4°C using the Intra Cellular Flow Cytometry kit (Cell Signaling Technology, MA, USA).

Techniques: Immunofluorescence, Flow Cytometry, Staining, Clone Assay, Derivative Assay, Infection