The following reagents and kits were used in this experiment: live/dead staining (#R37601, Invitrogen, Waltham, MA, USA), RPMI 1640 medium 1× (Cat. N.: SH30027.01, Cytiva, Tokyo, Japan), DPBS (Dulbecco’s phosphate buffered saline, ref. no. 21-031-CV, Corning Inc., Corning, NY, USA), DMEM/F-12 50/50, 1× (Dulbecco’s mod. eagle’s medium/Ham’s F-12 50/50 Mix, without L-glutamine, Corning, ref. no. 15-090-CV), FBS (fetal bovine serum, Cat. No.: S11150, Atlanta Biologicals®, Flowery Branch, GA, USA), pen/strep (penicillin–streptomycin, 10,000 units/mL penicillin, 10,000 µg/mL streptomycin, gibco®, ref. no. 15140-122), insulin (recombinant human insulin, gibco®, formula no. A11382IJ), UCG (Ultroser™ G, serum substitute for animal cell culture, PALL Life Sciences, 20 mL, ref. no. 15950-017), Exoview Kit, Avidin/Biotin Blocking Kit (SP-2001, Vector Labs, Burlingame, CA, USA), VECTASTAIN® ABC-HRP Kit, peroxidase (Vector Labs, PK-4000), DAB Substrate Kit, peroxidase (HRP), and nickel, (3,3′-diaminobenzidine) (Vector Labs, SK-4100).
Dulbecco phosphate buffered saline (dpbs)
Manufactured by Cytiva
Sourced in United States
DPBS, also known as Dulbecco's Phosphate Buffered Saline, is a balanced salt solution commonly used in cell culture applications. It provides a physiologically-compatible environment for maintaining and suspending cells in vitro. DPBS contains a mixture of salts, including sodium chloride, potassium chloride, sodium phosphate, and potassium phosphate, which help maintain osmotic balance and pH.
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Lab products found in correlation
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (2 mentions)
Live dead staining, by Thermo Fisher Scientific (1 mentions)
Dmem f12 50 50, by Corning (1 mentions)
Vectastain abc hrp kit, by Vector Laboratories (1 mentions)
Dab substrate kit, by Vector Laboratories (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Ultroser g, by Pall Corporation (1 mentions)
Lsrfortessa x 20 cell analyzer, by BD (1 mentions)
Annexin 5 binding buffer, by BioLegend (1 mentions)
Annexin 5 alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Zombie aqua, by BioLegend (1 mentions)
Accutase, by Thermo Fisher Scientific (1 mentions)
Optiprep, by Merck Group (1 mentions)
Sorvall legend rt centrifuge, by Thermo Fisher Scientific (1 mentions)
Tris buffered saline (tbs), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by R&D Systems (1 mentions)
M csf, by R&D Systems (1 mentions)
Rankl, by R&D Systems (1 mentions)
3 bromopyruvate, by Merck Group (1 mentions)
Vemurafenib, by Selleck Chemicals (1 mentions)
12 protocols using dulbecco phosphate buffered saline (dpbs)
1
Live/Dead Cell Staining Protocol
2
Isolation of Urine-Derived Stem Cells
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The Korean Public Institutional Review Board approved the collection of human urine samples in this study. In total, 200 mL urine was collected individually from healthy donors (n = 3) for the isolation of UDSCs. The collected urine was filtered using a 100 μm cell strainer (SPL, Pocheon, Korea) and then centrifuged at 500× g for 15 min. After removing the supernatant, the pellet was washed using DPBS (Cytiva, Marlborough, MA, USA) containing 1% antibiotic (Gibco, Waltham, MA, USA) and centrifuged at 500× g for 10 min (repeated 3 times). The cell pellets were suspended in growth media composed of KSFM and DMEM/F12 in a 1:1 ratio. The growth medium was supplemented with 5% FBS, 20 ng/mL EGF, 10 ng/mL bFGF, 300 ng/mL Hydrocortisone, 1% ITS-E, and 10 μM Y27632. The cells were seeded on a 24-well plate and cultured for 10–15 days at 37 °C. The growth medium was replaced every 2–3 days, and Y27632 was excluded. Colonies with rice grain-shaped cells were selectively collected and expanded after colony formation.
3
Fabrication of Collagen and PLA Scaffolds
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Collagen Scaffold Fabrication: Bovine collagen fibers (donation of Kaneka Corporation) were processed through a card chute system (Reiter Card C4), followed by two rounds of drawing (Reiter RSB851) until the fibers were anisotropically aligned. They were then passed through the roving machine (Reiter Fly F4/1) and then a ring spinning machine (Reiter G5/2), where yarns of 265 denier (yarn thickness unit) were produced [18 (link),19 (link)]. For this experiment, 12-inch pieces were cut and bundled up to make a tangled network of yarns. The samples were sterilized by soaking these yarns in a 70% ethanol solution and rinsing them with Dulbecco’s Phosphate-Buffered Saline (PBS) (DPBS, Cytiva). Each sample of yarn bundles was then placed into the wells of a 24-well plate. These yarn bundles are henceforth referred to as “collagen scaffolds”.
PLA Scaffold Fabrication: PLA fibers were provided by Xinxiang Sunshine Textiles Co., Ltd., Xinxiang, China, in yarn form (150 denier PLA yarn). The PLA yarn samples were spun into a yarn-like structure and processed with the same methods previously described for the collagen scaffold fabrication [19 (link)]. Twelve-inch pieces were cut and tangled into weblike bundles before being sterilized with 70% ethanol and washed with PBS. These weblike bundles are henceforth referred to as “PLA scaffolds”.
PLA Scaffold Fabrication: PLA fibers were provided by Xinxiang Sunshine Textiles Co., Ltd., Xinxiang, China, in yarn form (150 denier PLA yarn). The PLA yarn samples were spun into a yarn-like structure and processed with the same methods previously described for the collagen scaffold fabrication [19 (link)]. Twelve-inch pieces were cut and tangled into weblike bundles before being sterilized with 70% ethanol and washed with PBS. These weblike bundles are henceforth referred to as “PLA scaffolds”.
4
Bacterial Extracellular Vesicle Isolation
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Bacterial extracellular vesicles were generated as previously described with some modifications [7 (link)]. Briefly, bacteria were grown up to stationary phase in 120 mL of LB broth prior to being washed twice with 1× PBS and concentrated into 5 mL of PBS. The OD600 reading was taken to adjust the bacterial concentration to 108 cell/mL based on previous growth curves. Plating with serial dilution was used to confirm the bacterial count. The bacteria were then inoculated in two 60 mL flasks of fresh LB and grown for 12 h at 37 °C. The contents of each flask were centrifuged at 2000× g for 20 min at 4 °C to pellet out bacterial cells, after which the supernatant was ultracentrifuged at 25,000× g for 20 min at 4 °C. The resulting supernatants were filter sterilized with a 0.22 μm filter into new ultracentrifuge tubes and ultracentrifuged at 150,000× g for 2 h at 4 °C. The vesicle pellets resulting from the two flasks were resuspended in dPBS (Cytivia, Marlborough, MA, USA) and combined prior to a final ultracentrifugation spin at 150,000× g for 2 h at 4 °C before a final resuspension in 500 μL of diluent. Vesicles were stored at 4 °C before being used for quantification assays.
5
Apoptosis and Viability Assay for Endothelial Cells
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Protocol for assessment of apoptosis and viability and was adapted from reference 44 (link). Human umbilical vein endothelial cells were seeded at a cell density of 1 × 104 cells/cm2 in EMG2 + 10% media and allowed to adhere overnight. On the treatment day, cells were 80% confluent. Cells were washed with DPBS (Cytiva) and treated for 24 h with 10 μg/ml of purified IgG, pre-incubated with or without 1 μg/ml blocking peptide or human ACKR1 recombinant protein expressed with a proteo-liposome (H00002532-G01; Abnova) for 1 h at RT.
On the apoptotic assay day, cells were washed with DPBS and dissociated with accutase (Gibco) for 5 min. Dissociated cells were centrifuged for 5 min at 300g in DPBS. Cell pellets were washed once with Annexin V binding buffer (422201; BioLegend) and resuspended in Annexin V binding buffer containing antibody including Annexin V Alexa Fluor 488 (1:40, A13201; Invitrogen) and Zombie Aqua (1:200, 423101; BioLegend). Cells were stained in the dark for 15 min at RT. Stained cells were washed once with Annexin V binding buffer and resuspended in Annexin V binding buffer and analyzed using BD LSRFortessa X-20 Cell Analyzer (BD Biosciences) immediately. Data analysis was performed with Flowjo.
On the apoptotic assay day, cells were washed with DPBS and dissociated with accutase (Gibco) for 5 min. Dissociated cells were centrifuged for 5 min at 300g in DPBS. Cell pellets were washed once with Annexin V binding buffer (422201; BioLegend) and resuspended in Annexin V binding buffer containing antibody including Annexin V Alexa Fluor 488 (1:40, A13201; Invitrogen) and Zombie Aqua (1:200, 423101; BioLegend). Cells were stained in the dark for 15 min at RT. Stained cells were washed once with Annexin V binding buffer and resuspended in Annexin V binding buffer and analyzed using BD LSRFortessa X-20 Cell Analyzer (BD Biosciences) immediately. Data analysis was performed with Flowjo.
6
Virus-Like Particle Purification
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Five days post-transfection, cell suspensions (viability ≥95%) were centrifuged for 15 min at 3200 rpm on a Sorvall legend RT centrifuge (Thermo Fisher Scientific). Cell supernatants were then treated with 10 U/mL of Denarase (C-LEcta), 5 mM of MgCl2, and incubated for 1 h at 37 °C. For VLP sedimentation, treated supernatants were centrifuged over a 15% Optiprep (w/v) (Sigma-Aldrich)19 (link) cushion (10% of total volume) at 5300 × g for 16 h at 4 °C. After centrifugation, supernatants were discarded and pellets resuspended in DPBS (Cytiva).
7
ELISA Assay Buffer Optimization
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Assay buffers were screened and selected based on previous ELISA development by our laboratory (White et al., 2017 (link)). Initial experiments used a block and assay buffer consisting of 5% nonfat dried milk (Quality Biological), 0.5 mM Dulbecco's phosphate buffered saline (DPBS, Cytiva, formerly GE Healthcare Life Sciences), and 0.05% Tween® 20 (T-20, Fisher). Later experiments were conducted with a commercially available blocking buffer of 1% Hammarsten grade casein in tris(hydroxymethyl)aminomethane buffered saline (TBS, Thermo Scientific) with the addition of 0.05% T-20. Bridging studies were conducted to verify that changing the assay buffer had not impacted the previously identified assay conditions, as described below.
8
Osteoclastogenesis Regulation via BMP-2
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recombinant human bone morphogenetic protein-2 (rhBMP-2), Mouse-soluble receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) were purchased from R&D Systems (USA) and reconstituted in 0.1% bovine serum albumin that was dissolved in Dulbecco’s phosphate buffered saline (DPBS) according to the used dose. DPBS was purchased from Cytiva (USA). Trizol reagent was purchased from Invitrogen/Thermo Scientific (USA) ALP antibodies (AF2910) Antibody was purchased from R & D Systems. Antibodies for NFATc1 (sc-7294), c-Fos (sc-271243), Smad (sc-398844), Actin (sc-47778) and the Anti-mouse and rabbit antibody conjugated to horseradish peroxidase (HRP) were purchased from Santa Cruz Biotechnology (USA). All other antibodies were purchased from CST (USA).
9
Preparation of 3-Bromopyruvate and Vemurafenib
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3-bromopyruvate (Sigma Aldrich) was dissolved in DPBS (Cytiva, Marlborough, MA, USA) at room temperature immediately before use. Vemurafenib (Selleck Chem, Houston, TX, USA) was dissolved in dimethyl sulfoxide (DMSO; Sigma Aldrich) to prepare stock solutions of 10 µM, which were stored at 4 °C until use.
10
Quantifying Cell Apoptosis via Annexin V
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The viability of siRNA-treated cells (for 72 h) using the FITC Annexin V Apoptosis Detection Kit I (Cat No. 556547, BD Biosciences, San Diego, CA, USA) was performed, as suggested by the manufacturer. Briefly, all cells were mixed, washed with D-PBS (HyClone, Cytiva), resuspended in Binding Buffer at a final concentration of 106 cells/mL. Afterward, 100 µL (1 × 105) of cell suspension was probed with 5 µL of FITC-conjugated Annexin V and 5 µL of propidium iodide for 15 min protected from light. Then, 400 µL of Binding Buffer was added to each tube, and cells were analyzed by flow cytometry using a BD Accuri C6 Plus flow cytometer and dedicated BD Biosciences software (v.1.0.23.1; BD Biosciences).
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