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Adipogenesis differentiation kit

Manufactured by Thermo Fisher Scientific
Sourced in United States

The Adipogenesis Differentiation Kit is a laboratory tool designed to facilitate the in vitro differentiation of adipocytes from precursor cells. The kit provides a defined media formulation and optimized protocols to support the stepwise process of adipogenesis.

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10 protocols using adipogenesis differentiation kit

1

Multilineage Differentiation of Mesenchymal Stem Cells

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Cells (ovAD-MSCs, passage 3) were split and seeded onto 24-well plates (Corning) in DMEM2 at a concentration of 100,000 cells/mL (0.5 mL) and incubated until monolayers were 80% confluent. Media was changed to osteogenic, chorondrogenic or adipogenic differentiating medias (1 mL) (StemPro™ Osteogenesis Differentiation Kit, Adipogenesis Differentiation Kit, Chondrogenesis Differentiation Kit, Life technologies, Carlsbad, US). Cells were maintained for two weeks in the respective media, with media changed every 3 days. Differentiated cell monolayers were rinsed in DPBS (1 mL) then fixed in 10% neutral buffered formalin (1 mL) (Sigma-Aldrich) for 10 mins at rt°C. Monolayers of adipocytes were stained with Oil Red O (0.5% w/v isopropanol, 1 mL, rt°C, 10 min) (Sigma-Aldrich) and counter stained with 0.1% haemotoxylin (1 mL, rt°C, 10 min) (Sigma-Aldrich). Osteocytes were stained with 2% Alizarin Red S (1 mL, rt°C, 10 min) (Sigma-Aldrich). Chondrocytes were stained with Toluidine Blue (1 mL, 0.1% w/v, rt°C, 10 min) (Sigma-Aldrich). Stained monolayers were rinsed with ROH2O (3x, 1 mL) then imaged using an Olympus inverted phase contrast and microscope (IX51, Olympus, Tokyo, Japan).
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2

Multilineage Differentiation of muBM-MSCs

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muBM-MSCs were seeded onto Primaria™ 6-well plates and cultured in differentiating media (2 mL) (StemPro™ Osteogenesis Differentiation Kit, Adipogenesis Differentiation Kit, Chondrogenesis Differentiation Kit, Life technologies). For osteocyte and adipocyte differentiation cells were seeded at 80,000 per well, for chondrocyte differentiation cells were plated as 5 μL droplets of cell solution at a concentration of 160,000,000 cells /mL. Cells were cultured for 2–4 weeks before fixing with 4% paraformaldehyde (2 mL) (Sigma-Aldrich). Cultures were stained with either; 1% Lipidtoxgreen (2 mL) (Thermo Fisher Scientific, Waltham, Massachusetts), 2% Alizarin Red (2 mL) (Abcam, Milton, UK), 1% Alcian Blue (2 mL) (Sigma-Aldrich). Cultures rinsed with PBS (3x, 2 mL) then imaged at 100x using an Axiovert 35 inverted phase contrast and fluorescence microscope (Zeiss, Jena, Germany).
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3

Adipogenesis Differentiation Assay

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After the cells reached a proper confluence, the standard culture medium was changed to differentiation medium from the Adipogenesis Differentiation Kit (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). After 14 days of differentiation, cells were fixed with 4% PFA for 30 min and washed with PBS. After that, 60% isopropanol was added for 5 min. Staining was performed with 99% isopropanol and Oil Red O (Sigma-Aldrich, Saint Louis, MO, USA). The solution was diluted in distilled water (3:2). After removing isopropanol, the stain was added for 5 min in order to verify the positive effect of differentiation.
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4

Adipogenic Differentiation Assay

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When cells reached the proper confluence, the medium was replaced with differentiation medium from Adipogenesis Differentiation Kit (Gibco). After 14 days of differentiation, cells were fixed with 4% PFA for 30 minutes and washed with PBS and then 60% isopropanol was added for 5 minutes.
Staining was made of 99% isopropanol and Oil Red O (Sigma-Aldrich). The resulting solution was diluted in distilled water (3 : 2). Isopropanol was removed and the staining was added after 10 minutes of incubation on the cells for 5 minutes to verify positive effect of differentiation.
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5

Adipogenesis Differentiation and Staining

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When cells reached the proper confluence, the medium was replaced with differentiation medium from the Adipogenesis Differentiation Kit (Gibco, Thermo Fisher Scientific, Waltham, MA, USA).). After 14 days of differentiation, cells were fixed with 4% PFA for 30 min and washed with PBS, and then 60% isopropanol was added for 5 min.
Staining was achieved with 99% isopropanol plus Oil Red O (Sigma-Aldrich, Saint Louis, MO, USA). The resulting solution was diluted in distilled water (3:2). Isopropanol was removed and the stain added after 10 min of incubation of the cells for 5 min to verify the positive effect of differentiation.
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6

Multilineage Differentiation of Stem Cells

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After expansion in the mini-bioreactor, cells were evaluated regarding their potential to differentiate into adipocytes, osteocytes and chondrocytes. For adipogenic and osteogenic differentiation, cells were plated in duplicate at a density of 3000 cells/cm2 on a 24-well plate (pre-coated with CELLstart, dilution 1:100) with culture medium. At confluence, cells were induced to differentiate into osteocytes and adipocytes using StemProTM Osteogenesis and Adipogenesis Differentiation Kit (Gibco, Grand Island, NY, United States), respectively. Culture medium was exchanged twice a week. Adipogenesis was observed after 14 days for lipid droplets (Sudan II-Scarlet) and osteogenesis after 21 days for mineralized bone matrix deposition (von Kossa). For chondrogenic differentiation, 1 × 107 cells were plated in droplets (5 μL) on Ultra-Low attachment multi- well plates (Corning, Lowell, United States). The plate was left in the incubator for 30 min and afterward StemProTM Chondrogenesis Differentiation medium (Gibco, Grand Island, NY, United States) was added. Total medium exchange was performed two times a week for 15 days. Cells were then stained with Alcian blue (1%, Sigma- Aldrich, St Louis, MO, United States) for 2 h to assess proteoglycan synthesis (Mizukami et al., 2016 (link)).
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7

Multilineage Differentiation of Mouse MSCs

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Balb/c mice (2–3 weeks old) were decapitated, and the femur and tibia were carefully removed. Bone marrow cells were flushed with a syringe (27‐gauge) inserted into one end of the bone and cultured at a density of 3 × 105 cell/cm2 with low‐glucose Dulbecco's modified Eagle's medium (DMEM; Gibco) containing 15% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Gibco). After 2 hours, nonadherent cells were removed via medium changes, which were repeated every 3 days until the cultured cells reached 70%–80% confluence. Cells were then digested with TrypLE Express (Gibco) for 2 minutes and passaged. All experiments used MSCs at passage 5–8.
To evaluate their multipotent differentiation potential, MSCs were incubated at passage 5 in a 12‐well plate at a density of 5 × 103 cells/cm2 with osteogenesis differentiation medium (Gibco) or at a density of 1 × 104 cells/cm2 with an adipogenesis differentiation kit (Gibco) for 3 weeks. According to the manufacturer's instructions, other MSCs were seeded at a higher density of 1 × 107 cells per milliliter to formulate a micromass and cultured with a STEMPRO chondrogenesis differentiation kit (Gibco) with medium changes every 3 days for 2 weeks. Osteoblasts were stained using Alizarin Red S (Sigma), adipocytes were stained with Oil Red O (Sigma), and chondrogenesis pellets were stained with Toluidine Blue (Sigma).
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8

Adipogenesis Differentiation of MSCs

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At passage three, human BM-derived MSCs were cultured with the Adipogenesis Differentiation Kit (Gibco, USA) according to the manufacturer’s instructions. At day 14 of differentiation, oil red O staining was carried out. Briefly, cells were washed with PBS and fixed in 4% paraformaldehyde for 30 minutes. After washing twice with PBS, cells were stained with 0.3% oil red O solution (Sigma Aldrich, USA) for 20 minutes at room temperature. Staining was quantified by extracting oil red O from the stained cells with isopropanol, followed by determining the optical density (OD) values of the solution at 518 nm. The NAC treatment method was performed according to the method published by Tormos et al [13 (link)]. The NAC treatment was started on Day 2 of differentiation and lasted until Day 14. On Day 1, the differentiation and NAC+differentiation groups were treated with the Adipogenesis Differentiation Kit, while the undifferentiated and NAC-treated groups were not subjected to adipocyte differentiation. On Day 2, cells were exposed for 4 hours prior to ROS measurement to NAC treatment (5 mM) or to PBS for control group.
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9

Multilineage Differentiation of UC-MSCs

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Adipogenic and osteogenic differentiation assays at the fourth passage were conducted to detect the multilineage differentiation potential of the isolated human UC‐MSCs. UC‐MSCs were induced to differentiate using an Adipogenesis Differentiation Kit (A1007001, Gibco) and Osteogenesis Differentiation Kit (A1007201, Gibco). Lipid accumulation was detected at 7–14 days after induction culture by Oil Red O staining. For osteogenic differentiation, the calcium deposition was detected at 21–28 days after induction culture by Alizarin Red S staining.
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10

Multilineage Differentiation of Bcl-2-ADSCs

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To induce adipogenic differentiation, Bcl-2-ADSCs were seeded onto plates at 1 × 10 4 cells/cm 2 in complete DMEM. After culturing for 24 h, the medium was changed to adipogenic induction medium using an Adipogenesis Differentiation Kit (Gibco) and the cells were cultured for 14 days. Then, Oil Red O staining was used to observe oil droplets in the cytoplasm of cells. For chondrogenic differentiation, 5-μL droplets of Bcl-2-ADSCs in complete DMEM (1 × 10 7 cells/mL) were seeded onto plates. After culturing for 48 h, the medium was changed to chondrogenic induction medium using a Chondrogenesis Differentiation Kit (Gibco). Chondrogenic differentiation was induced for 14 days. Chondrogenic differentiation was assessed using Alcian Blue staining. For osteogenic differentiation, Bcl-2-ADSCs (5 × 10 3 /cm 2 ) were seeded onto plates in complete DMEM. After culturing for 48 h, the medium was changed to osteogenic induction medium using an Osteogenesis Differentiation Kit (Gibco) and cells were cultured for 24 days. The cells were then stained with a 1% Alizarin Red S solution.
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