Commercialized human umbilical vein endothelial cells (HUVECs) (BCRC) were utilized for the current study. Cells were first grown at 37°C in a 100 mm culture dish (Falcon) in M199 (Gibco) medium with endothelial cell growth supplement, heparin, and 20% fetal bovine serum (FBS) (Gibco). Cells of the third to fifth generation were used in the current study (Additional file 1 ).
100 mm culture dish
Manufactured by BD
Sourced in Morocco
The 100 mm culture dish is a circular, flat-bottomed container made of plastic or glass. It is designed to provide a sterile environment for the growth and cultivation of cells, tissues, or microorganisms in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Mouse multipotent mesenchymal stromal cell marker antibody panel, by R&D Systems (1 mentions)
Wnt5a, by R&D Systems (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Model 680, by Bio-Rad (1 mentions)
4 protocols using 100 mm culture dish
1
Culturing Human Umbilical Vein Endothelial Cells
2
Isolation and Culture of Murine Mesenchymal Stem Cells
3
Tyrosinase Activity Assay in B16 Melanoma Cells
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Tyrosinase activity was determined according to a previously reported method (Lee et al. 2015) (link) with slight modifications. B16 melanoma 4A5 cells were seeded at 3.0 × 10 6 cells/dish in a 100-mm culture dish (BD Falcon) and precultured at 37 °C for 24 h. After removing culture media, 10 mL of fresh DMEM containing 10% FBS, 100 nM α-MSH, and 1.6 mM L-tyrosine were added to the dish and cultured at 37 °C for 72 h. After removing culture media, cells were washed with PBS once. After removing PBS, cells were suspended in 2 mL of 10 mM sodium phosphate buffer (NaPB, pH 7.4) containing 0.01% Triton-X 100 and 50 nM phenylmethylsulfonyl fluoride and frozen at -35 °C. After thawing the cells, the suspension was centrifuged at 13,000 rpm for 10 min at 4 °C, and the supernatant was used as a tyrosinase solution.
Every reaction of tyrosinase activity assay consisted of 50 μL of 2 mM L-dopa and 100 μL of 10 mM NaPB containing 1 μL of SME, 1 μL of 100 mM arbutin, or 1 μL of DMSO alone (control) in each well of a 96-well microplate. After preincubation at 37 °C for 10 min, 50 μL of the tyrosinase solution (0.7 mg protein/mL) was added to each well of the microplate and incubated at 37 °C for 60 min. The produced amount of dopachrome was determined by measuring the absorbance at 450 nm using a microplate reader (Model 680, Bio-Rad Laboratories).
Every reaction of tyrosinase activity assay consisted of 50 μL of 2 mM L-dopa and 100 μL of 10 mM NaPB containing 1 μL of SME, 1 μL of 100 mM arbutin, or 1 μL of DMSO alone (control) in each well of a 96-well microplate. After preincubation at 37 °C for 10 min, 50 μL of the tyrosinase solution (0.7 mg protein/mL) was added to each well of the microplate and incubated at 37 °C for 60 min. The produced amount of dopachrome was determined by measuring the absorbance at 450 nm using a microplate reader (Model 680, Bio-Rad Laboratories).
4
Isolation and Purification of BMSC and HSC
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BMSC and HSC were isolated from SD rats of each group. Rats were gently flushed out of tibias and femurs using F-12K and 10 % FBS. Cells obtained from two tibias and two femurs were plated in 100 mm culture dish (BD Falcon) in 10 ml of BMSC media at 37°C with 5 % CO 2 . After 24 h, non-adherent cells were collected for purification of CD34 + HSC. The remaining adherent cells, which were abundant in MSC, were trypsinised and replated until they achieved >85 % density. Non-adherent cells were incubated with CD34-conjugated magnetic microbeads (Miltenyi Biotec) according to the manufacturer's instructions. Afterwards, CD34 + populations were isolated by using a separation column placed in a magnetic field, and were then subjected to phenotypic and functional assays with more than 90 % survival rate determined by trypan blue staining.
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