Ultrafiltration centrifuge tube

PTD is a short peptide (YGRKKRRQRRR) that mediates protein entry into cells.²⁴ (link) The PTD-ATF6 or PPM1H coding sequence was inserted into pET-28a+ plasmids. After transfer into E. coli strain Transetta (DE3) (Transgen Biotech, China), the recombinant proteins were induced by isopropyl β-D-thiogalactoside (IPTG), and the supernatant was subjected to Ni affinity chromatography after ultrasonic lysis of the bacterial solution (6×His-Tagged Protein Purification Kit – Soluble Protein, CWBIO). Then proteins were concentrated using an ultrafiltration centrifuge tube (Millipore) with a filter pore size of 30 kDa. The purified His-PPM1H was used to perform the in vitro phosphatase assay. PTD-ATF6 was applied to incubate cells at a certain concentration, and the culture medium was changed after 48 h.

The EE% was determined by centrifugation method (Lv et al., 2020 ). Briefly, 500 μL of HAS-NLCs was taken into the ultrafiltration centrifuge tube (0.5 mL, 100 kDa, Millipore, Billerica, MA) and centrifuged at 3000 rpm for 15 min. Then, the filtrate from tubes was collected, diluted with methanol to 5 mL and analyzed by HPLC. Besides, equal volumes of HAS-NLCs were broken, diluted to 5 mL with methanol and analyzed by HPLC. The EE% of HAS-NLCs was calculated by the following equation (Moghddam et al., 2016 ):
$$\text{EE}\%=\frac{W_{\text{total}}-W_{\text{free}}}{W_{\text{total}}}\times 100\%$$
where EE% is the entrapment efficiency of HAS-NLCs; W_total is the total amount of HAS and W_free is the amount of HAS-free in HAS-NLCs; their units are mg.
The recovery experiment of EE% was conducted by standard procedures and elaborated in the supplementary material.
The DL% was measured by HPLC. Ten milligrams of freeze-dried HAS-NLCs powders were thoroughly dissolved with methanol, diluted to 5 mL and determined by HPLC. The DL% of HAS-NLCs was calculated by the following equation (Lakhani et al., 2019 ):
$$\text{DL}\%=\frac{W_{\text{total}}-W_{\text{free}}}{W}\times 100\%$$
where DL% is drug loading of HAS-NLCs; W_total is the total amount of HAS and W_free is the amount of HAS-free in HAS-NLCs; W is the weight of freeze-dried HAS-NLCs; their units are mg.

The spores were extensively washed with distilled water and then decoated with the alkaline decoating method [37 (link)]. Briefly, the spore coat layer was stripped with a detergent solution consisting of 0.1 M NaOH, 0.1 M NaCl, 0.1 M Dithiothreitol (DTT) and 0.5% sodium dodecyl sulfate (SDS). The stripping process involved incubating spores in 1 mL of the detergent solution at 70°C for 30 min. Then the residual spores were removed by centrifugation, and the supernatant was dialyzed against deionized water for 24 h in an ice bath. After dialysis, the supernatant was concentrated using an ultrafiltration centrifuge tube (Millipore, Bedford, MA). A blend of the concentrated supernatant and loading buffer (62.5 mM Tris-HCl, 10% glycerol, 2% SDS, 1% 2-mercaptoethanol, 0.003% bromophenol blue, pH 6.8) was boiled for 5 min. The boiled solution (approximately 50 μL) was analyzed with 12% SDS-PAGE and stained with Coomassie brilliant blue R-250. The clear bands were cut horizontally into slices (1.5 mm wide) for MALDI-TOF MS analysis. Dithiothreitol and 2-mercaptoethanol were both purchased from Sigma-Aldrich (Shanghai, China), and the rest of the ingredients were purchased from Sinopharm (Shanghai, China).

According to the methods of Torreggiani et al. [20 (link)], PRP samples were centrifuged at 250 g for 15 min to obtain PRP microspheres and the platelet pellet was washed with PBS (Ca²⁺-free and Mg²⁺-free, Gibco). After activating 4 ml PRP suspension with 1 ml of 10% CaCl₂ and 1000 U thrombin (Hunan Yige Pharmaceutical, China), the suspension was centrifuged in series at low speeds (300 g for 10 min, 2000 g for 10 min) to discard cell debris. Then, the supernatant was filtered through a 0.22 μm filter (Millipore, Germany), and the filtrate was transferred to a 15 ml ultrafiltration centrifuge tube (Millipore) under 4000 g centrifugation for 50 min. The liquid was washed with PBS and ultrafiltered at 4000 g again. To purify the Exo, the medium was added onto a 30% sucrose/D₂O cushion in an Ultra-ClearTM tube (Beckman Coulter, USA) and ultra-centrifuged at 100,000 g for 70 min. After washing by PBS, Exo suspension was ultracentrifuged again at the same high speed for 70 min. The Exo were then carefully resuspended in sterile PBS and stored at − 80 °C for subsequent experiments.

The N-terminal of mouse CYLC1 was cloned into the pET-N-His-C-His vector (Beyotime, Shanghai, China) and then transfected into the ER2566 E. coli strain (Weidi Biotechnology, Shanghai, China). CYLC1 protein expression was induced by 1 mM IPTG (Beyotime) at 24°C for 6 hr. After centrifugation, the bacterial pellet was resuspended in buffer 1 (50 mM Tris–HCl pH 8.0, 200 mM NaCl), and the proteins were released by sonication. After centrifugation, anti-His beads (Beyotime) were added to the supernatant and incubated for 2 hr. After washing five times with buffer 1, CYLC1-N protein was eluted with 250 mM imidazole (Beyotime). The protein was concentrated by centrifugation with ultrafiltration centrifuge tube (Millipore, Shanghai, China). One hundred micrograms of CYLC1-N protein was emulsified at a 1:1 ratio (vol/vol) with Freund’s complete adjuvant (Beyotime) and administered subcutaneously into New Zealand white rabbits (Charles River) at multiple points. For the subsequent three immunizations, 50 μg CYLC1-N protein was emulsified with incomplete Freund’s adjuvant (Beyotime) at an interval of 2 weeks. One week after the last immunization, blood was collected, and the serum was separated.

Compound 8 (20 mg) was dissolved in DMF (150 μL) in a micro-centrifuge tube, and then triethylamine (8–10 μL) was added. The color of the solution turned yellow. Nonasaccharide (3 mg) was dissolved in DMF (150 μL) in a micro-centrifuge tube, and then it was slowly added to the solution of compound 8. The micro-centrifuge tube was washed for dissolving the nonasaccharide with DMF (100 μL), and then it was also added to the reaction mixture and mixed well. The reaction mixture was stirred for 4 h at room temperature. The reaction solution was transferred to a micro-thick-walled reaction vial, and then it was concentrated under reduced pressure and further dried under vacuum. After that, the residue was dissolved in water, and it was washed with CH₂Cl₂. The aqueous phase was evaporated at room temperature. The residue was dissolved in 0.01 M PBS buffer (600 μL, pH 7.6), and a 0.01 M PBS solution of protein CRM197 (400 μL, 10 mg/mL) was added. Then, the solution was rotated at room temperature without mechanical stirring for 24 h. After that, the solution was centrifuged (4 °C, 12,000 g) with an ultrafiltration centrifuge tube (Millipore^®, 0.5 mL, 30 kD) for 5 min and washed three times with ultrapure water. The remaining samples in the ultrafiltration centrifuge tube were diluted and taken out to obtain an aqueous solution containing CRM197 glycoconjugates.