Coomassie plus bradford assay

For studies of RH1 reduction, cells were grown in 22 cm² cell culture dishes. Cells were scraped off and suspended in 500 μL of buffer containing 25mM Tris-HCl (pH 7.4) and 250mM sucrose. Cells were disrupted with an ultrasonic homogenizer (Bandelin Sonopuls HD 2070, Berlin, Germany) and protein concentration was measured with a Coomassie Plus Bradford Assay (Thermo Scientific, Vilnius, Lithuania). Cell lysates were diluted to a concentration of 1 mg/ml with the same buffer which has been used for cell disruption. The RH1 reduction rate was measured in the presence of the NADPH regeneration system or by adding 500 μM NMeH (reduced N-methylnicotinamide, Santa Cruz Biotechnology, Heidelberg, Germany). For the NADPH regeneration system we used 20 μM NADPH, 10 mM glucose-6-phosphate and 10 µ/mL glucose-6-phosphate dehydrogenase. The reactions were followed spectrometrically using a Hitachi 557 UV-Vis spectrometer at 37 °C. The RH1 reduction was monitored at 328 nm with the NADPH regeneration system or at 318 nm (NMeH isosbestic point) when NMeH was used as a cosubstrate. NMeH oxidation was monitored at 380 nm. Spectral data was first processed with R Studio and then statistical analysis was performed with MS Excel software.

The hESC‐MSC EVs were prepared as previously described (Lai et al., 2016). Briefly, immortalized hES‐ MSCs were grown in a chemically defined medium for 3 days and CM was harvested and pre‐cleared with a 0.22 µm syringe filter. The CM was concentrated 100× for exosomes by tangential flow filtration (Sartorius; MWCO 100 kDa) and stored in −20°C freezer until use. The EVs were assayed for protein concentration using Coomassie Plus (Bradford) Assay (Thermo Fisher Scientific) per manufacturer's instruction and characterized for particle size distribution and concentration by Zetaview (Particle Metrix) according to the manufacturer's protocol. Follow‐up experiments from subsequent batches of hESC‐MSC EVs (posterior to batch AC83) yielded identical results.

E. coli cells were transformed with either pET30-ctbciA or pET30-rsbciA, and single colonies were used to inoculate 1 L LB broth supplemented with kanamycin (30 µg/ml). Protein expression was induced at an OD₆₀₀ of 0.6 upon addition of isopropyl β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM. The culture was incubated for an additional 16 hours at 30°C and cells were harvested by centrifugation. The cell pellet was resuspended in Buffer A (50 mM Tris-HCl, 100 mM NaCl, 5 mM imidazole, pH 8.0) and cells were lysed by sonication. The supernatant was clarified by centrifugation at 15 000×g for 20 minutes and the soluble portion was loaded onto a 5 ml HisTrap FF column (GE Healthcare, Piscataway, NJ) which was equilibrated with Buffer A. The protein was eluted from the column over a linear gradient to 100% Buffer B (50 mM Tris-HCl, 100 mM NaCl, 250 mM imidazole, pH 8.0) over 20 column volumes. The resulting fractions were analyzed by SDS-PAGE to assess protein purity. Fractions determined to be >95% pure were pooled and the protein concentration was determined by Coomassie Plus Bradford assay (Thermo Scientific, Rockford, IL).