Centricon centrifugal filter devices

The pAAV-EF1a-R-CaMP1.07 construct was obtained by subcloning the R-CaMP1.07 ORF from the original plasmid pN1-R-CaMP1.07 [7 (link)] into a AAV-EF1a-DIO-EYFP target vector (Addgene #27056) at BamHI and EcoRI sites. Recombinant serotype 1 AAV particles were produced in adherent AAV-293 cells by co-transfection with the pDF1 helper plasmid, using standard procedures. AAV1 particles were isolated from cell lysates using an iodixanol density gradient followed by anion exchange FPLC (GE Healthcare Bio-Sciences AB). Vector suspensions were concentrated in PBS using Centricon^® centrifugal filter devices with a molecular weight cut-off of 100 kDa (Millipore, Billerica, MA). For titration, the number of encapsidated genomes per ml was determined by real-time PCR.

CS-NPs was prepared by ionotropic gelation method. Briefly, chitosan, 0.1 g, was dissolved in 100 mL acetic acid (1 mol/L) with continuous stirring and followed by adding 1 mol/L NaOH to get the final pH of the resulting mixture around 6.5. Then, the appropriate volumes of TPP (1 mg/mL) was slowly added dropwise into the prepared chitosan solution while stirring in room temperature (25 °C) for 1 h to obtain CS-NPs; the ratio of chitosan and TPP was 4:1. Further, the reaction mixture was then centrifuged twice at 10,000× g for 20 min with discarding the supernatant to collect the formed CS-NPs. The technique of carbodiimide chemistry was used for the formation of Lys-CS-NPs. Next, 10 mg/mL of lysozyme was mixed with 0.1 M EDAC and 0.1 M NHS to form the activated ester, and then the activated lysozyme was added into the CS-NPs at a final concentration of 0.05-0.50 mg/mL to obtain the Lys-CS-NPs. Finally, the reaction mixture was sonicated under ultrasonic conditions at 24 kHz, 8 W power, and 6 min with Ultrasonic Cell Disruption System (JY92-IIN, Ningbo, China) and ultrafiltrated with the Centricon centrifugal filter devices (molecular weight cut-off 50 kDa, Millipore, Darmstadt, Germany).

Cell-bound and shed forms of syndecan-1 were quantified using the CD138 ELISA Kit (Diaclone Research, Besancon, France) according to the instructions of the manufacturer. Conditioned, serum-free media were concentrated using Centricon centrifugal filter devices with 10 kDa nominal molecular weight limit filters (Merck Millipore, Burlington, MA, USA). Cells were lysed in a buffer containing 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 0.5% v/v Triton X-100, and protease inhibitor cocktail (Sigma-Aldrich). The protein content of the samples was determined by Coomassie reagent (Bio-Rad Laboratories Inc.) and 300 μg total protein from each sample was applied to the ELISA plate. Optical densities were read at 450 nm in a LabSystems Multiskan MS microplate reader (Thermo Fisher Scientific).