Figure 3

Analysis of the purified LVs. Cell culture SN was purified by UC or MA/UF. The concentrates were analyzed by electron microscopy: 40 ml SN, MA/UF (A); 500 ml SN, MA/UF (B); 500 ml SN, UC (C). (D) Analysis of biological vs. physical titer (IP/VP) of different LV preparations: The biological titer was determined via FACS-analysis of transduced cells and the physical titer by use of a HIV-1 p24 Antigen ELISA (p24 ELISA) or QRTPCR (Realtime). Values were determined in triplet and/or use of different dilutions. Concentrates of the following purifications were analyzed: 500 ml SN, MA/UF (n = 7); 500 ml SN, UC (n = 3); 40 ml SN, MA/UF (n = 4); 40 ml SN, UC (n = 7). mean+SEM. * 0.05 ≥ p > 0.01; *** 0.001 ≥ p; ns - not significant. (E), (F) Analysis of protein and DNA content of LV preparations. After transfection of producer cells the medium was changed to 40 ml DMEM, supplemented with FCS (DMEM) or to serumfree medium Panserin 608 (Pans) or HyClone SFM4Megavir (SFM). Purification of LVs was performed by MA/UF. 10 μl of SN or concentrate (conc) as well as 5 μg pure BSA was loaded on the SDS-PAgel (coomassie blue stain). M, protein marker (E). 500 ml of the same cell culture SN was each incubated at 37°C for 30 min either without addition of benzonase (-benz) or after adding 12.5 u/ml benzonase (+benz). The SN was purified using MA/UF, respectively. 5 μl of the elution fraction (el) or the concentrate (conc) were used for agarose gel electrophoresis. M, DNA marker (F).