Journal: Biomédica

Article Title: Assessing the oncolytic potential of rotavirus on mouse myeloma cell line Sp2/0-Ag14

doi: 10.7705/biomedica.4916

Figure Lengend Snippet: Infection of Sp2/0-Ag14 cells by rotavirus isolates and infectious virions present in supernatant from the culture medium. Sp2/0-Ag cells at the logarithmic growth phase were separately infected with previously trypsin- activated rotavirus isolates WTEW, WWM, Wt1-5, TRUYO, or ECwt-O at 0.8 MOI each. A. HRP immunochemistry staining of rotavirus structural antigens (reddish) at 12 hpi followed by FITC fluorescent staining (green). B. Sp2/0-Ag cells were separately infected with the rotavirus isolates mentioned above and cell aliquots taken at the indicated post-infection times. Infection was recorded as the percentage of cells being positive to rotavirus structural antigens using HRP immunochemistry assay. C. As in B, except that cells being positive to nonstructural antigen NSP 4 were recorded. D. As in B, except that cells being positive to non-structural antigen NSP5 were recorded. E. Control non-tumoral L929 cells were separately infected with the rotavirus isolates indicated above using increasing MOI. Infection is expressed as the percentage of cells being positive to rotavirus structural antigens at 12 hpi using HRP immunochemistry assay. F. As in E, except that control cells consisting of non-tumoral PBMCs were used instead of L926 cells. G. Sp2/0-Ag cells were infected as described in A and then aliquots of cells were collected every 2 h after infection with the indicated rotavirus isolates and subjected to centrifugation at 700 g . The supernatant medium was collected and used to inoculate a fresh batch of Sp2/0-Ag cells, which were collected at 12 hpi and lysed; the infectious titers (FFU/ml) of lysates were determined by immunochemistry assay for the viral structural antigens. H. As in G, except that the non-structural antigen NSP4 was determined. I. As in G, except that the non-structural antigen NSP5 was determined. Infection is expressed as mean percentage ± SD from three independent experiments (n=3), each performed in duplicate.

Article Snippet: Donkey anti-goat or anti-rabbit antibodies conjugated with FITC (SC-362255 and SC-362261, respectively) or HRP (SC-2020 and SC-2313, respectively) were also obtained from Santa Cruz Biotechnology Inc. 7-aminoactinomycin D (7-AAD), propidium iodide, and 4,’6-diamidino-2-phenylindole (DAPI) were purchased from Invitrogen (Carlsbad, CA, USA).

Techniques: Infection, Staining, Control, Centrifugation

Journal: Biomédica

Article Title: Assessing the oncolytic potential of rotavirus on mouse myeloma cell line Sp2/0-Ag14

doi: 10.7705/biomedica.4916

Figure Lengend Snippet: Inhibition of rotavirus infection by antibodies against cell surface proteins. A. Dot plots of fluorescent events in Sp2/0-Ag cells labeled by FITC-conjugated antibodies to the indicted cell surface proteins. Positive cells were selected by gating FITC-labelled cells in a FITC vs side scatter plot. B-K. Sp2/0-Ag cells were incubated with serial dilutions of antibodies against the indicated cell surface proteins or the indicated synthetic peptides. Cells were separately challenged with rotavirus isolates ECwt-O (B, C), TRUYO (D, E), Wt1-5 (F, G), WTEW (H, I) or WWM (J, K) at MOI 0.8 each. Percentage of infection Inhibition at 12 hpi in terms of cells positive to rotavirus structural antigens was expressed relative to the corresponding rotavirus-infected control cells that had not been antibody-treated. Infected and non-infected cells treated with unrelated isotype-matched antibodies were used as a control. Percentages were normalized to those of the infected cells that had been treated with isotype antibody. L. Representative images from an immunochemistry assay of cells non-infected (left panel), infected with virus isolate WTEW (central panel), and pre-treated with hyperimmune antiserum (reciprocal dilution 40 x 103) against Hsp90 (right panel) before infection with isolate WTEW.

Article Snippet: Donkey anti-goat or anti-rabbit antibodies conjugated with FITC (SC-362255 and SC-362261, respectively) or HRP (SC-2020 and SC-2313, respectively) were also obtained from Santa Cruz Biotechnology Inc. 7-aminoactinomycin D (7-AAD), propidium iodide, and 4,’6-diamidino-2-phenylindole (DAPI) were purchased from Invitrogen (Carlsbad, CA, USA).

Techniques: Inhibition, Infection, Labeling, Incubation, Control, Virus

Journal: Biomédica

Article Title: Assessing the oncolytic potential of rotavirus on mouse myeloma cell line Sp2/0-Ag14

doi: 10.7705/biomedica.4916

Figure Lengend Snippet: Effect of ECwt-O infection on the expression of cell surface proteins in Sp2/o-Ag cells. Cells were inoculated with ECwt-O at MOI 2. Confocal microscopy was used to determine expression changes of the indicted cellular proteins at 1 and 12 hpi. Alexa fluor 568 (red) and FITC (green)-labelled secondary antibodies were used to visualize cellular proteins and rotavirus structural proteins, respectively. Nuclei were counterstained with DAPI (blue). Cells were analyzed using a confocal microscope (Nikon C-1) and analyzed using the EZ-C1, ver. 3.90, software.

Article Snippet: Donkey anti-goat or anti-rabbit antibodies conjugated with FITC (SC-362255 and SC-362261, respectively) or HRP (SC-2020 and SC-2313, respectively) were also obtained from Santa Cruz Biotechnology Inc. 7-aminoactinomycin D (7-AAD), propidium iodide, and 4,’6-diamidino-2-phenylindole (DAPI) were purchased from Invitrogen (Carlsbad, CA, USA).

Techniques: Infection, Expressing, Confocal Microscopy, Microscopy, Software