Infinite pro nanoquant

The determination of total AAV particle concentration (TP) was carried out with a conformational AAV2 ELISA assay (Progen Biotechnik GMBH, Heidelberg, Germany) according to the manufacturer’s instructions. The reference curve was built from 2.3 × 10 ${}^9$ TP mL ${}^{-1}$ with serial dilutions to 1.8 × 10 ${}^7$ TP mL ${}^{-1}$ . The absorbance was quantified at 450 nm on an Infinite PRO NanoQuant (Tecan, Männedorf, Switzerland) microplate multimode reader using a clear 96-plate well provided in the kit. The samples were applied at multiple dilutions.

The cells were harvested, counted, pelleted at 300 g for 10 min. Cell lyses was performed using Mammalian Protein Extraction Reagent (M-PER) (Thermo Scientific™) with 1x cOmplete™ EDTA-free Protease Inhibitor Cocktail (Roche Applied Science), using 100 μL per 2 million of cells. The mixture was vortexed and placed at 4 °C for 10 min. Extracts were clarified by centrifugation (14.000× g for 10 min). Samples were stored at −20 °C for short-term and −80 °C for long-term. The total protein content was quantified using a BCA Protein Assay Kit (Thermo Fisher Scientific). Samples were applied in serial dilutions and reference material was applied in duplicate. The absorbance was measured with an Infinite PRO NanoQuant (Tecan) microplate reader.

Total particle (TP) quantification was performed with conformational AAV8 ELISA XPRES kit (PROGEN) according to the manufacturer’s instruction. Samples were diluted in working buffer and applied in triplicate. Absorbance measurements were obtained at 450 nm, using 650 nm as a reference, on Infinite^® PRO NanoQuant (Tecan) microplate reader.
Viral genome (VG) copies were quantified by qPCR. DNA was extracted using the High Pure Viral Nucleic Acid Kit (Roche). qPCR was performed with a probe (5′-TTG​CCG​TCC​TCC​TTG​AAG​TCG​AT-3′) and transgene-specific primers (forward primer, 5′-GAA​CCG​CAT​CGA​GCT​GAA-3′ and reverse primer, 5′-TGC​TTG​TCG​GCC​ATG​ATA​TAG-3′). The quantification was performed using the LightCycler system (Roche Diagnostic) using the eGFP transgene plasmid as standard.