Dulbecco s phosphate buffered saline

Fresh porcine flank skin pieces were supplied from a local butcher’s shop and were transferred with ice to the laboratory and were kept frozen in −20 °C refrigerator. On the experiment day, the skin was thawed at room temperature. Upon after, the skin was cut into pieces with appropriate sizes to be put on the device. The insertion of MNAs into the skin was performed using Instron ElectroPuls Model E10000 Axial Torsion Test System (Illinois Tool Works Inc., Glenview, IL, USA) in compression mode with precise measurement of the required force for the skin perforation. Custom holders were fabricated using 3D printing for placing the skin samples onto the shafts of the device. Data was collected every 20 ms. Furthermore, in order to keep the skin fresh and prevent its dehydration during experiment, skin samples were kept on paper tissues soaked with liquid Dulbecco’s phosphate-buffered saline w/o calcium w/o magnesium (Biowest, Lakewood Ranch, FL, USA).

Food wheat starch (Tibet İthalat İhracat ve Kozmetik, Istanbul, Türkiye) in two values of 30 g and 15 g were added to two beakers with 75 mL distilled water. Both mixtures were put on a magnetic hot plate stirrer (Hangzhou Miu Instruments, Hangzhou, China) at 100 °C and 400 rpm stirring, for 24 min. The solution with 15 g starch was chosen as the model drug carrier. 0.075 g RhB (REF 1.07599.0025, Sigma Aldrich, St. Louis, MO, USA) as the model drug was added to the starch solution and was completely stirred to result a uniform solution. Tips of an example MNA (case 6 in Table 1, featuring a designed based diameter and height of 1500 μm and 2000 μm, respectively, and draft angle of 5°, etched in a 5 M KOH solution for 14 h) were coated with the prepared solution and were inserted into the porcine skin with hand. In order to keep the skin fresh and prevent its dehydration during experiment, skin sample was kept on paper tissues soaked with liquid Dulbecco’s phosphate-buffered saline w/o calcium w/o magnesium (Biowest, Lakewood Ranch, FL, USA). After removing the MNA from the skin, the perforation site was imaged under visible light, and under ultraviolet (UV) light (OmniCure S2000, Excelitas Technologies, Waltham, MA, USA).

BUT, IPA, histamine, ionomycin, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′- tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), trypsin-EDTA solution, NaCl, KCl, MgCl₂, CaCl₂, glucose, and 4-(2-hydroxyethyl)-1-piperazineethane- sulfonic acid (HEPES) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Dulbecco’s Phosphate Buffered Saline was obtained from Biowest (Nuaillé, France). Astrocyte Medium, Astrocyte Growth Supplement, fetal bovine serum (FBS), penicillin/streptomycin solution, and poly-L-lysine were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA).

Roswell Park Memorial Institute (RPMI) 1640 medium was obtained from Gibco-Life Technologies (USA). Fetal bovine serum (FBS), penicillin–streptomycin mixture (Biowest, France) and Dulbecco’s phosphate buffered saline (DPBS) were purchased from Biowest (Nuaille’, France). Blood cell lysis buffer, amphotericin B, trypan blue, concanavalin A (ConA) and lipopolysaccharide (LPS) were obtained from Sigma–Aldrich (USA).

Nitroblue Tetrazolium (NBT) assay was performed after 4 days of drugs treatment. Following treatment with different compounds, cells were collected, centrifuged at 300 × g for 7 minutes at room temperature and resuspended in complete IMDM growth medium or in alternative in RPMI growth medium at the density of 3.0 × 10⁵ cells/mL with NBT (Nitro blue Tetrazolium Chloride) (Sigma-Aldrich, #N6876) 1 mg/mL and MA (Phorbol 12-myristaPte 13-acetate) 5 µg/mL (Selleckem, # S7791). Next cells were incubated for 60 min at 37 °C. In differentiated cells NBT is phagosomed, the intracellular enzymes convert NBT into insoluble blue formazan crystals. At the end of the incubation cells were collected, centrifuged at 300 × g for 7 min and washed in PBS (Dulbecco’s Phosphate Buffered Saline, Biowest, #L0625-500). Next cells were resuspended in complete IMDM growth medium or in alternative in RPMI growth medium and seeded on 12 well plate (Falcon®, #353043). For each sample images were acquired by light microscopy using an inverted microscope (Nikon, model Eclipse Ts2 #136710). At least 200 cells for sample were counted using ImageJ software and the percentage of differentiated cells (cells containing blue-black formazan deposits) was calculated and processed using GraphPad Prism 7 software.