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. 2012 Jun 8;23(10):1031–1042. doi: 10.1089/hum.2012.066

FIG. 1.

FIG. 1.

AAV1, AAV2, and AAV3b exhibit different transduction profiles in 911 cells in the presence of increasing amounts of soluble heparin. (A) Direct comparison of luciferase expression between serotypes AAV1, AAV2, and AAV3b in response to increasing concentrations of soluble heparin. 911 cells were transduced with 10,000 particles per cell of AAV1 or AAV3 and only 1000 particles per cell of AAV2, all bearing the luciferase transgene. All transduction experiments described were performed in the absence of an adenoviral helper to eliminate compounding effects of a second virus. The use of 10 times more AAV1 and AAV3 in this experiment compared with AAV2 was done to obtain reliable signals and is consistent with work performed previously. The vectors were applied to 911 cells in a 96-well format. Infections were performed in the presence of increasing amounts of heparin ranging from 0 to 1000 μg/ml in 2-fold serial dilutions. Light emitted from the infections was assayed by luminometer the next day. These experiments in 911 cells were repeated a total of three times and data similar to that shown in Fig. 1 were observed. Data represent means±SD. (B) Response of AAV3b daughter vector, SASTG, to soluble heparin. Four independently generated preparations of the SASTG-luciferase vector gave similar results in the presence of increasing amounts of heparin in 911 cells. SASTG vectors (1000 particles/cell) were applied to 911 cells (10,000 per well) in a 96-well format. Infections were performed in the presence of increasing amounts of heparin ranging from 0 to 1000 μg/ml in 2-fold serial dilutions. Light emitted from the infections was assayed by luminometer the next day. Data represent means±SD. (C) FACS assay demonstrating the alteration of SASTG GFP expression in 911 cells with low concentrations of heparin. Infections were performed in the presence of heparin (1.95 or 3.9 μg/ml). GFP-positive cells were counted 5 days postinfection via flow cytometry. Similar results were observed in three independent experiments. Data are presented as percent GFP-positive cells and represent means±SD.