The MAC-EV treatment increased the hair growth in individual #1 (Figure 8A). from scalp skin after informed consent was obtained Amitriptyline HCl from the patients. The study was approved by the Medical Ethical Committee of Kyungpook National University and Hospital (Daegu, Republic of Korea) and was conducted in accordance to principles and guidelines of the Declaration of Helsinki. DP cells were isolated from the bulbs of dissected hair follicles, transferred to tissue culture dishes coated with bovine type I collagen, and cultured in DMEM low-glucose (HyClone, Logan, UT, USA) supplemented with 1??Antibiotic-Antimycotic, 1?ng/mL bovine Amitriptyline HCl fibroblast growth factor, and 20% heat inactivated FBS at 37?C. The explants were cultured for 7 days, and the medium was changed every 3 days. The isolated DP cells were then plated in 100 mm culture dishes containing DMEM low-glucose, supplemented with 10% heat-inactivated FBS. The cells were sub-cultured according to the percentage of confluence, and cell passage number 2 2 was used in this study . 2.3. Isolation of Extracellular Vesicles and Condition Media for Macrophages When the cells were about 80% confluent, extracellular vesicles Amitriptyline HCl were extracted from the culture media of macrophages using ultracentrifugation, as described previously with modification . Briefly, the medium was centrifuged at 1500 for 10 min, at 2000 for 20 min, and then at 10,000 for 30 min, at 4 C, to remove the unwanted cells and debris. Next, the supernatant was filtered through a 0.45 m pore size filter. A small portion of the medium was collected, called Amitriptyline HCl EV-media (EV-M; media containing EVs), and stored at ?80 until experimental use. This medium was then ultra-centrifuged at 100,000 for 60 min, and the supernatant was collected, called EV-depleted media (EV-DM; media containing no EVs), and stored at ?80 . The EV pellets were washed with phosphate-buffer saline (PBS) by ultracentrifugation, as stated above, reconstituted with 50C100 L PBS, and stored at ?80 . The ultracentrifugation was performed using Mouse monoclonal to CD29.4As216 reacts with 130 kDa integrin b1, which has a broad tissue distribution. It is expressed on lympnocytes, monocytes and weakly on granulovytes, but not on erythrocytes. On T cells, CD29 is more highly expressed on memory cells than naive cells. Integrin chain b asociated with integrin a subunits 1-6 ( CD49a-f) to form CD49/CD29 heterodimers that are involved in cell-cell and cell-matrix adhesion.It has been reported that CD29 is a critical molecule for embryogenesis and development. It also essential to the differentiation of hematopoietic stem cells and associated with tumor progression and metastasis.This clone is cross reactive with non-human primate a SW28 rotor, and ultra-clear tubes of optima TML-100 XP ultracentrifuge (Beckman Coulter, GA, USA). The EV concentrations were measured by Pierce Bicinchonic Acid Protein Assay Kit (Thermo Fisher Scientific, MA, USA) and represented as its total protein concentration (per mL) in this study. 2.4. Western Blot Analysis Western blot analysis was performed as described in a previous study . Whole cells and EV-lysates were prepared in Sodium Dodecyl Sulfate (SDS) lysis buffer (62.5 mM Tris, pH 6.8, 2% SDS, 0.1% -mercaptoethanol, 10% glycerol, and protease inhibitor cocktail (Sigma, MO, USA). Equal amounts of protein were loaded and separated using 10% SDS- polyacrylamide gel electrophoresis. The proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA), probed first with the primary antibody, and then with the secondary antibody conjugated with horseradish peroxidase (see Supplementary Table S1 for details). The signals were detected using enhanced chemiluminescence (GE Healthcare, IL, USA) according to the manufacturers instructions. Blot images were cropped and prepared using Picasa3 (version 220.127.116.11.1) (Google, CA, USA) and/or PowerPoint program (Microsoft, WA, USA) (contrast was adjusted, if necessary, for better visualization). Band intensity was measured by GelQuant.NET software (Version 1.8.2) (BiochemLabSolutions.com, CA, USA). 2.5. Transmission Electron Microscopy (TEM) The MAC-EVs pellets were resuspended in 100 L of 2% paraformaldehyde. Next, 5 L EVs pellets were attached to the Formvar-carbon coated with EM grids, and covered with protective material like aluminum foil for 20 min to avoid any damage/dryness to the sample. About 100 L of PBS was added on a sheet of parafilm and grids were transferred on to the Amitriptyline HCl drops of PBS, using sterile forceps for washing. Next, it was transferred to 50 L of 1% of glutaraldehyde and incubated at 25C30 for 5 min, and then washed with distilled water for 2 min. Samples were stained using 2% uranyl acetate. These steps were repeated 7 more times, and samples were allowed to completely dry before observing under an HT 7700 transmission electron microscope (Hitachi, Tokyo, Japan) to view the size of the EVs . 2.6. Nanoparticle Tracking Analysis (NTA) The measurement of size of MAC-EVs was performed by Nano Sight LM 10 (Malvern, Worcestershire, UK) according to the instructions provided. The sample was diluted 1000-folds in milli-Q water, a sterile syringe was used to inject the sample into the chamber, and.
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