ADSCs in passages 4-6 were used to collect conditioned medium. For the hypoxic group, ADSCs that were 80 - 90% confluent were starved with serum-free DMEM/F12 medium at 37°C in an incubator with 1% oxygen and 5% CO2 for 48h. For the normoxic group, ADSCs were incubated in an incubator with 5% CO2. Then, the ADSCs supernatant was collected, centrifuged at 3500 rpm for 10 minutes, and filtered using a 0.22 μm Millex GP syringe filter (Millipore, USA) to ensure sterility. The product is ADSC-CM. After that, ADSC-CM was transferred to an ultrafiltration centrifugal tube(Millipore, USA, UFC9003) and centrifuged at 3500 rpm for 25 minutes. The upper product used as cADSCs-CM was collected and stored at -80°C until use. For convenience, in this study, the cADSC-CM obtained in the hypoxic environment was called hypo-CM, and the one obtained in the normoxic environment was called nor-CM.
Ultrafiltration centrifugal tube
Manufactured by Merck Group
Sourced in United States
The Ultrafiltration centrifugal tube is a laboratory equipment designed for the concentration and purification of macromolecules, such as proteins, enzymes, and nucleic acids, through the process of ultrafiltration. It utilizes centrifugal force to separate the desired molecules from the solution based on their molecular weight and size.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Oregon green 488, by Thermo Fisher Scientific (2 mentions)
Axio observer 7 microscope, by Zeiss (2 mentions)
Millex gp, by Merck Group (1 mentions)
Tecnai g2 tem, by Thermo Fisher Scientific (1 mentions)
Xl100k ultracentrifuge, by Beckman Coulter (1 mentions)
0.22 μm filter, by Merck Group (1 mentions)
1 2 dipalmitoyl sn glycero 3 phosphocholine dppc, by Merck Group (1 mentions)
Ni nta his bind resin, by Merck Group (1 mentions)
Rpmi 1640, by Sartorius (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Papain, by Yuanye Bio-Technology (1 mentions)
Ammonium formate, by Thermo Fisher Scientific (1 mentions)
Pectinase, by Yuanye Bio-Technology (1 mentions)
Formic acid, by Thermo Fisher Scientific (1 mentions)
Methanol, by Thermo Fisher Scientific (1 mentions)
Dab peroxidase substrate kit, by Solarbio (1 mentions)
Cellulase, by Yuanye Bio-Technology (1 mentions)
Hplc grade acetonitrile, by Thermo Fisher Scientific (1 mentions)
Ovalbumin (ova), by Merck Group (1 mentions)
12 protocols using ultrafiltration centrifugal tube
1
Hypoxic ADSC Conditioned Medium Preparation
2
Acid-Responsive Nanogels for Drug Delivery
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Acid-responsive nanogels were prepared using the UV-crosslinking method. Briefly, the nanogels were obtained by dissolving the polymer of PVA-g-VEA-DMA/TPP in water (1 mg/ml) with the addition of a photoinitiator (I2959, 5 wt% of polymer), adjusting solution pH to 6.8, and stirring under UV exposure for 10 min. To prepare drug-loaded nanogels, a solution of PTX and LND in DMSO (10 mg/ml) was added in the aqueous polymer solution with a feeding ratio of 10%. Through adjusting the solution pH to 6.8 and in situ crosslinking of acrylate in the polymers under UV light for 10 min, PTX and LND co-loaded nanogels were obtained. A Millipore ultrafiltration centrifugal tube with a MWCO of 10,000 was used to remove the free drugs.
3
Exosome Isolation and Characterization Protocol
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The supernatants were collected to isolate exosomes via ultracentrifugation according to a previous study [52 (link)]. The supernatants were centrifuged at 800 g for 15 min followed by 10,000 rpm for 30 min at 4 °C to remove cells and debris and then ultrafilter and concentrated at 2000 rpm for 20 min using ultrafiltration centrifugal tube (Millipore, USA). Concentrated supernatants were centrifuged at 140,000g for 90 min at 4 °C in a Type Ti100 rotor using an XL-100K ultracentrifuge (Beckman). After resuspension in PBS, the exosome pellet was ultracentrifuged again for 90 min at 140,000g. Finally, the exosomes were resuspended in PBS, filtered using a 0.22-μm filter (Millipore, USA), and analyzed with a Enhance BCA Protein Assay kit (Beyotime, China). Approximately 6 mg of exosomes are obtained per 500 ml of supernatant. The purified exosomes were resuspended in PBS (200 µl) and further diluted by 1- to 10- hundred folds for analysis. The samples were fixed with 2.5% glutaraldehyde overnight at 4 °C. Ten microliters of the mixture were applied to copper grids and stained with 1% phosphotungstic acid for 1 min. The dried grid was observed by a Tecnai G2 TEM (FEI, USA). NTA was conducted using a Zeta View system (NanoSight, UK) to automatically track the Brownian motion and size distribution data of exosomes in real time.
4
Purification of ApoA-I Protein Variants
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The 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and the PMSF were purchased from Sigma–Aldrich (St. Louis, MO, U.S.A.); the Pierce BCA Protein Assay kit was purchased from Thermo Fisher Scientific (Rockford, IL, U.S.A.). Ni-NTA His Bind resin and ultrafiltration centrifugal tube were purchased from Millipore (Billerica, MA, U.S.A.); RPMI-1640 and Certified were purchased from Biological Industries (Kibbutz Beit Haemek, Israel). Human THP-1 monocytes and recombinant Escherichia coli containing the coding region for the human ApoA-Iwt and cysteine mutants were preserved in the Laboratory of The Affiliated Hospital of Qingdao University. All other chemical reagents were obtained commercially and were of analytical grade.
5
Immunogenic Ovalbumin with Alum Adjuvant
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ImjectTM Alum Adjuvant (No. 77161) was obtained from Thermo Scientific Inc. (Pittsburgh, PA, USA). Ovalbumin (OVA) was obtained from Sigma-Aldrich Inc. (Saint Louis, MO, USA). BCA protein assay kit was obtained from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Goat anti-mouse IgE antibody and HRP-labeled donkey anti-goat IgG antibody were obtained from Abcam Inc. (Cambridge, UK). DAB peroxidase substrate kit was purchased from Solarbio Co., Ltd. (China). The HPLC-grade acetonitrile, methanol, ammonium formate and formic acid were obtained from Fisher Scientific (Pittsburgh, PA, USA). Ultrapure water was collected from Millipore Milli-Q system (Bedford, MA, USA). Cellulase (400 U/mg), pectinase (500 U/mg) and papain (800 U/mg) were obtained from Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Ultrafiltration centrifugal tube (15 mL, 10 kDa) was purchased from Millipore Inc. (Bedford, MA, USA). Other reagents were purchased from Sigma-Aldrich Inc. (Saint Louis, MO, USA).
6
SARS-CoV-2 N protein phase separation
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The dimeric N constructs with removed cellular nucleic acids were labeled by fluorescence dye Oregon-Green488 (Invitrogen, 2,161,802) with a molar ratio 10:1 between protein and fluorescence dye in labeling buffer 50 mM NaPhosphate pH 7.0, 50 mM NaCl. The mixtures were incubated at 25 ℃ for 1 h. To remove the excess fluorescence dye, the resulting mixture was buffer-exchanged with labeling buffer using concentrator columns with 10 kDa cutoff (Ultrafiltration Centrifugal Tube, Millipore). The SARS-CoV-2 5' UTR and 3' UTR were labeled by fluorescence dye Cy3 (Lumiprobe, #41,070) following the Cy3 labelling protocol [79 (link)]. The labeled proteins and RNAs were quantified using a NanoDrop OneC (Thermo Scientific).
Oregon-Green488 labeled N constructs were diluted to a final concentration of 25 μM in phase separation buffer (50 mM Tris–HCl pH 7.5, 100 mM NaCl) containing 10% PEG 3350, and RNAs were added with a protein to RNA molar ratio of 100:1. Then the mixture was incubated at room temperature for 5 min. A total of 10 μL solution was transferred onto the glass slide, and images were collected using a Zeiss-Axio Observer 7 microscope.
Oregon-Green488 labeled N constructs were diluted to a final concentration of 25 μM in phase separation buffer (50 mM Tris–HCl pH 7.5, 100 mM NaCl) containing 10% PEG 3350, and RNAs were added with a protein to RNA molar ratio of 100:1. Then the mixture was incubated at room temperature for 5 min. A total of 10 μL solution was transferred onto the glass slide, and images were collected using a Zeiss-Axio Observer 7 microscope.
7
Characterization and Encapsulation Efficiency of Transfersomes
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The particle size, PDI, and zeta potential of transfersomes and CPP-modified transfersomes were determined at 25 ℃ using particle size and zeta potential analyzer Nanotrac Wave II (Microtrac, Largo, FL, USA). The morphology of LR-loaded CPP-modified transfersomes was visualized using TEM (HT7700; Hitachi, Tokyo, Japan). The encapsulation efficiency (EE) of various transfersome formulations was determined by ultrafiltration centrifugation. Freshly prepared (0.4 mL) LR@STFs, LR@DTFs, LR@STFs-CPP, or LR@DTFs-CPP (LR 1 mg/mL) was added into the ultrafiltration centrifugal tube (100 kDa, Millipore) and centrifuged at 6000 × g for 15 min. The collected filtrate and original formulation were diluted in ethanol with 1% acetic acid. For the quantitative determination of LR, its fluorescence intensity (λex/λem = 285/370 nm) was measured using a microplate reader (SpectraMax M2; Molecular Devices, Sunnyvale, CA, USA). The EE of LR in different formulations was calculated according to the following equation: where Wt is the quantity of LR in the unfiltered formulation, and Wf is the quantity of LR in the filtering medium.
8
JWYPFS-Medicated Serum Preparation Protocol
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JWYPFS-medicated serum was prepared based on previous reports (Xu et al., 2020 (link)). Ten adult male SD rats (weight 200–250 g) were provided by Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). After 1 week of adaptive feeding, the rats were randomly divided into two groups; the normal group was given saline, while the second group was treated with JWYPFS by oral gavage (0.96 g/ml, 1 ml/100 g body weight), twice a day for seven consecutive days. Two hours after the last administration, the rats were anesthetized by intraperitoneal injection of pentobarbital sodium (30 mg/kg). Blood was collected from the heart of the rat. The blood samples were left to stand 1 h and then centrifuged at 3500 rpm for 10 min, filtered with an Ultrafiltration centrifugal tube (Millipore), heated in 56°C water bath for 30 min, and stored at −80°C.
9
Pyroptosis Activation and Protein Isolation
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BMDMs were cultured in a 10-cm cell culture dish 24 h before the stimulation. Cells were treated with 20-ng/mL TNFα and 10-μg/ml CHX for 6 h to induce pyroptosis activation. Approximately 1 × 107 cells were harvested and washed three times with 4 °C PBS. Then, cells were resuspended in 1 ml of PBS containing 1 × protease inhibitor cocktail. Ultrasonic disruption was performed using a sonicator with the following parameters: 20 kHz and 3 min (six cycles of 30s on, 30 s off) on ice. Following sonication, cell lysates were centrifuged at 14,000×g for 10 min at 4 °C. The resulting supernatants were collected for further concentration. Then, 200 μL of the cell lysate was loaded into the ultrafiltration centrifugal tube (Millipore, 10kd) and centrifuged at 4000×g for 15 min at 4 °C until the volume was reduced to approximately 20 μL.
10
Formulation and Characterization of PTX-Loaded RGD-Targeted Liposomes
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Egg distearoylphosphatidylcholine (EPC) was sourced from Lipoid GMBH (Germany). DSPE-PEG2000 and DSPE-PEG2000-RGD were purchased from Shanghai Ponsure Biotech, Inc. (Shanghai, China). Cholesterol (Chol) was provided by Shanghai Xingzhi Chemical Factory (Shanghai, China). PTX was obtained from Wuhan bio-cata Biological Technology Co., Ltd (Wuhan, China). Coumarin-6 (C6, purity > 98%) was purchased from Sigma Aldrich (St. Louis, USA). 1,1’-dioctadecyl-3, 3, 3’, 3’-tetramethylindotricarbocyanine iodide (DiR) dye was obtained from Yeasen Biotechnology (Shanghai, China). DMEM medium was supplied by Gibco (CA, USA). Fetal bovine serum (FBS) was sourced from GEMINI (USA). The high-performance liquid chromatography (HPLC)-grade solvents used for HPLC were obtained from Xiqiao Chemical Co., Ltd. (Shantou, China). Other chemicals and reagents were obtained commercially and were of analytical grade. Ultrafiltration centrifugal tube (molecular weight cut-off: 10,000) were supplied from Millipore (Massachusetts, USA). DSPE-PEG2000-LHRHa (LHRHa peptides with amino acid sequence:17 (link) His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-Gly-Cys) were synthesized by ChinaPeptides Co., Ltd (Shanghai, China), with the Time-of-Flight Mass Spectrometry results shown in Figure S1
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