Cell suspension at passage 4 was seeded in six-well plates with a concentration of 8 × 103 cells/cm2 in a ready-made chondrogenic induction medium (Gibco StemPro Thermo Fisher Scientific, Massachusetts, USA); the medium was changed 2 times per week for 28 days. After that time, the cell cultures were washed twice by PBS, fixed by 4% paraformaldehyde for 1 hour, washed twice by distilled water, then stained in dark with Alcian blue (10 mg in 60 ml ethanol with 40 ml acetic acid) overnight; the next day, the cell culture was destained by 120 ml ethanol with 80 ml acetic acid for 20 minutes, finally washed 2 times by PBS, and then checked under a microscope (Figure 2 ).
Gibco stempro
Manufactured by Thermo Fisher Scientific
Sourced in United States
Gibco StemPro is a specialized cell culture medium designed to support the growth and maintenance of human pluripotent stem cells. It provides a defined, serum-free, and xeno-free environment for the cultivation of embryonic stem cells and induced pluripotent stem cells.
Automatically generated - may contain errors
4 protocols using gibco stempro
1
Chondrogenic Differentiation of Passage 4 Cells
2
Osteogenic Differentiation Assay
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Cell suspension at passage 4 was seeded in six-well plates with a concentration of 8 × 103 cells/cm2 in a ready-made osteogenic induction medium (Gibco StemPro, Thermo Fisher Scientific, Massachusetts, USA), according to Gronthos et al.'s protocol; the medium was changed every 3 days for 28 days in humidified incubators ([19 (link), 20 (link)]), the cell cultures were washed twice by PBS, fixed by 4% paraformaldehyde for 1 hour, washed again twice by distilled water, finally stained by 2% Alizarin Red for 45 minutes, finally washed 4 times by distilled water and 2 times by PBS, and checked under a microscope (Figure 2 ).
3
Adipogenic Differentiation of Stem Cells
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Cell suspension at passage 4 was seeded in six-well plates with a concentration of 8 × 103 cells/cm2 in a ready-made adipogenic induction medium (Gibco StemPro, Thermo Fisher Scientific, Massachusetts, USA) according to Pittenger et al.'s protocol; the medium was changed 2 times per week for 28 days [21 (link)]. After that time, the cell cultures were washed twice with PBS, fixed by 4% paraformaldehyde for 1 hour, and washed again twice by distilled water; the cell culture is washed by 60% isopropanol for 5 minutes, then stained for 5 minutes by Oil Red O in isopropanol (300 mg Oil Red in 100 ml isopropanol), washed by tap water, and finally stained by hematoxylin for 1 minute, again washed by tap water, and checked under a phase-contrast microscope (Figure 2 ).
4
Isolation and Maturation of Human Mast Cells
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Buffy coats were purchased from the blood donation center (Swiss Red Cross, Bern, Switzerland). The study was approved by the local ethics committee. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation on Ficoll Paque (GE Healthcare). The general protocol for generating blood-derived human mast cells is published elsewhere (15) . Here, mast cell progenitors were isolated using CD133 microbeads (Miltenyi Biotec) and in vitro matured over 8 weeks. The cells were cultured in Gibco™ StemPro (Thermo Fisher Scientific) containing supplements, 100ng/ml Gibco™ stem cell factor (Thermo Fisher Scientific) and 40 ng/ml IL-6 (Biolegend). Additionally, the medium contained 10 ng/ml IL-3 (Biolegend) for the first 3 weeks and after 6 weeks, the medium was supplemented with 10 ng/ml IL-4 (Peprotech, Rocky Hill, NJ, USA) and 10% FCS.
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