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. 2016 Jun 15;7(30):47287–47301. doi: 10.18632/oncotarget.10075

Figure 2. Chemical property analysis and biological activity of recombinant Ad vectors.

Figure 2

(A) rAd5-zTRAIL-RFP (A1) and rAd5pz-zTRAIL-RFP (A2) purified by a second round of CsCl continuous density gradient centrifugation. The Ads are located in the white bands. (B) Immunogold labeling of rAd5-zTRAIL-RFP and rAd5pz-zTRAIL-RFP viruses reacted with anti-rabbit gold-coupled IgG with or without primary anti-TRAIL antibody. The rAd5-zTRAIL-RFP virus was incubated with the anti-TRAIL antibody and anti-rabbit IgG antibody coupled to 6-nm gold particles (top row), rAd5pz-zTRAIL-RFP was incubated with both primary and secondary antibodies (bottom row). A rAd5pz-zTRAIL-RFP control sample was reacted only with the secondary antibody (middle row). (C) The content of TRAIL on the modified virus surface was detected by ELISA. **P < 0.01. (D) The physicochemical properties of purified virus include physical titers and infectious titers, and ratios among them, and TRAIL content. (E) Apoptosis of ZR-75-30 cells induced by different MOIs and times were determined by fluorescence microscopy using the Annexin-V/PI reagent kit. Fluorescence images at 400× magnification showed changes of recombinant Ad-infected tumor cells stained with Annexin-V/PI. (F) ZR-75-30 cells treated with two viruses (rAd5-zTRAIL-RFP, rAd5pz-zTRAIL-RFP) at 100 MOI for 2, 4, 6 and 8 h. The cells were analyzed using the apoptosis assay kit and flow cytometry.