Amicon ultra 0.5 centrifugal filter

The alginate, P407, and pNIPAAM solutions were prepared by dissolving each polymer in a citrate–phosphate solution with the last two in an ice bath. Polymer was gradually added under magnetic stirring until apparent solution homogeneity was achieved. The final solutions were kept at 4 °C for 48 h under slight magnetic stirring until the polymer fully dissolved. The concentrations of the pNIPAAM and P407 solutions were 10 and 250 mg/mL, respectively. The sodium alginate was dissolved in a citrate–phosphate solution at 40 °C. Alginate was added to the citrate–phosphate solution under gentle magnetic stirring. The resulting 10 mg/mL solutions were then magnetically stirred at room temperature overnight. Finally, the pNIPAAM, alginate and P407 solutions were kept at 4 °C for 24 h. The sdAb solutions were concentrated up to 6.5 mg/mL using the Amicon^® Ultra-0.5 centrifugal filter from Sigma-Aldrich (Saint-Quentin-Fallavier, France). The sdAb and polymer solutions were mixed at a ratio of 1:4 (v/v). The resulting concentrations of sdAb, pNIPAAM, alginate, and P407 were 1.5, 8, 8, and 200 mg/mL, respectively. To improve the solutions’ homogeneity, they were mixed by means of multiple pipetting, left at 4 °C for 24 h, pipetted a second time, and then stored at 4 °C.

The sdAbs were concentrated to 3 mg/mL using the Amicon^® Ultra-0.5 centrifugal filter from Sigma-Aldrich (Saint-Quentin-Fallavier, France) and then dialyzed at 4 °C against the citrate–phosphate solutions using the Pierce™ 96-well Microdialysis Plate (3.5K MWCO) from Thermo Fisher Scientific (Illkirch-Graffenstaden, France). The ratio of protein to buffer was 1:16 (v/v), at 100 µL of protein solution to 1.6 mL of dialysis buffer. The dialysis buffer was exchanged 3, 6, 16, 19, 22, and 32 h after dialysis began. The protein solutions and dialysis buffers were recovered and sterile-filtered under a laminar flow cabinet using 0.22 µm polyethersulfone filters from Sigma-Aldrich (Saint-Quentin-Fallavier, France). The sdAb solutions were centrifuged for 20 min at 20,000 g and 4 °C and then sterile-filtered as previously described. Protein concentration was determined by measuring absorption at 280 nm using a NanoDrop^TM 100 from Thermo Fisher Scientific (Illkirch-Graffenstaden, France). The protein solutions were adjusted to 0.5 and 0.2 mg/mL for subsequent biophysical studies.

Preparation of TP-Exos was conducted according to the literature [26 (link)] with some modifications. Namely, 3 mg (protein concentration) of purified exosomes dispersed in 900 μl PBS (pH 7.2 - 7.4) was mixed with 100 μg TP dissolved in 100 μl DMSO, and then the mixture was sonicated by an ultrasonic cell crusher (JY92-II, China) (20% power, 15 cycles of a 2 s pulse/2 s pause). After sonication, the mixture was incubated at 37°C for 60 min to allow for recovery of the exosomal membrane. Excess free TP and DMSO were separated from the mixture by centrifugation at 12,000 × g for 30 min using the Amicon Ultra-0.5 Centrifugal Filter (10 kDa, Millipore) and then washed twice with precooled PBS. Finally, the pellet was suspended in PBS, passed through a 0.22 μm syringe filter for sterility, and stored at –80°C until use for studies.

NF-kB activation was assessed in 6 days differentiated astrocyte-like cells (see above), stimulated, 45 min before being collected, with 10 ng/ml of IL1β in fresh medium.
Proteins were collected by using an extraction kit (Active Motif, Catalog # 40010), according to manufacturer’s instruction. The whole-cell lysates from mNPsc were concentrated by means of Amicon Ultra-0.5 centrifugal filter devices (Millipore) to achieve the recommended concentration of 20 μg of protein per well per sample (20 μl volume). The protein concentration was determined before and after concentration, by the BCA protein assay (Pierce).
NF-kB activation in whole-cell lysates was determined by the TransAM NFkB p65 kit (Active Motif, Catalog # 40096), following the manufacturer’s instructions. This 96 well ELISA-based kit provides colorimetric readout of NF-kB p65 subunit activation, quantified by spectrophotometry at the wavelength of 450 nm (reference wavelength 655 nm). As a positive control for p65 activation, Jurkart nuclear extracts, provided with the kit, were used. Detection limit: <1 μg lysate/well.