Oil red O staining (Solarbio) was performed to investigate the effect of ethanol (100 mM), alda‐1 (20 μM) and the AMPK antagonis DMP (10 μM) on the adipogenic differentiation of BMSCs. In briefly, each well of six‐well plates was seeded with 2 × 105 cells in 2 ml medium. Then adipogenic medium (Cyagen, China) was added to every well for inducing adipogenic differentiation when the confluence of cells was 80%, and the adipogenic medium A and B were refreshed by the protocol. Oil red staining was performed at day 21.
Adipogenic medium
Manufactured by Cyagen
Sourced in United States, China
Adipogenic medium is a cell culture medium designed to promote the differentiation of cells into adipocytes (fat cells). It contains a specific combination of growth factors, hormones, and other supplements that are necessary for the adipogenic differentiation process.
Automatically generated - may contain errors
Lab products found in correlation
Chondrogenic medium, by Cyagen (6 mentions)
Osteogenic medium, by Cyagen (6 mentions)
Ab81289, by Abcam (3 mentions)
Mir 342 3p agomir, by Sangon (3 mentions)
Mir 342 3p antagomir, by Sangon (3 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
Inverted microscope, by Zeiss (2 mentions)
Oil red o staining, by Cyagen (1 mentions)
Fluorescence inversion microscope system, by Zeiss (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Six well plate, by Corning (1 mentions)
Alizarin red staining, by Cyagen (1 mentions)
Ab181597, by Abcam (1 mentions)
Chondrogenic differentiation medium, by Cyagen (1 mentions)
Oil red o staining, by Merck Group (1 mentions)
Cetylpyridinium chloride, by Merck Group (1 mentions)
Gelatin from porcine skin, by Merck Group (1 mentions)
Collagenase type 1, by Merck Group (1 mentions)
Alpha minimum essential medium, by Thermo Fisher Scientific (1 mentions)
25 protocols using adipogenic medium
1
Adipogenesis Modulation by Ethanol and AMPK
2
Osteogenic and Adipogenic Differentiation Assay
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The cells (2 × 105 cells/well) were loaded in six-well plates (Corning, USA). Once the cells reached 80% confluence, the medium was changed to commercial osteogenic medium (Cyagen Biosciences, China) or adipogenic medium (Cyagen Biosciences, China). After 14 days of induction, the cells were fixed in 4% paraformaldehyde (Sigma-Aldrich, USA) for 30 min and then subjected to Alizarin Red staining (Cyagen Biosciences, China) to reveal calcium depositions or Oil Red O staining (Cyagen Biosciences, China) for the observation of lipid droplets. The cells were imaged with a Fluorescence Inversion Microscope System (Carl Zeiss, Germany).
3
Multilineage Differentiation of Red Panda eMSCs
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For adipocytic differentiation, eMSCs at passage 4 were seeded in 6-well plates and treated with adipogenic medium (Cyagen) as per the manufacturer’s protocol. The medium was changed three times per week. After 8 days, adipogenesis was evaluated by Oil red O staining (Sigma). Staining was assessed by bright-field inverted microscopy (IX73, Olympus). Red panda eMSCs cultured in normal growth medium served as control.
For chondrogenic differentiation, eMSCs at passage 4 at a density of 4 × 105 cells were cultured in chondrogenic differentiation medium (Cyagen) as per the manufacturer’s protocol. The medium was changed three times per week. After 21 days, chondrogenesis was detected by the staining of toluidine blue. Red panda eMSCs cultured in normal growth medium served as control.
For hepatogenic differentiation, eMSCs were cultured in hepatogenic differentiation medium (Cyagen) as per the manufacturer’s protocol. The medium was changed three times per week. After 16 days, hepatogenic differentiation was evaluated by cytokeratin 18 (CK18) (ab181597, Abcam) immunofluorescence staining, detection of CK 18, ALB and DKK1 mRNA expression, ALB protein expression, Periodic Acid-Schiff (PAS) staining and Indocyanine Green (ICG) uptake. Red panda eMSCs cultured in normal growth medium served as control.
For chondrogenic differentiation, eMSCs at passage 4 at a density of 4 × 105 cells were cultured in chondrogenic differentiation medium (Cyagen) as per the manufacturer’s protocol. The medium was changed three times per week. After 21 days, chondrogenesis was detected by the staining of toluidine blue. Red panda eMSCs cultured in normal growth medium served as control.
For hepatogenic differentiation, eMSCs were cultured in hepatogenic differentiation medium (Cyagen) as per the manufacturer’s protocol. The medium was changed three times per week. After 16 days, hepatogenic differentiation was evaluated by cytokeratin 18 (CK18) (ab181597, Abcam) immunofluorescence staining, detection of CK 18, ALB and DKK1 mRNA expression, ALB protein expression, Periodic Acid-Schiff (PAS) staining and Indocyanine Green (ICG) uptake. Red panda eMSCs cultured in normal growth medium served as control.
4
Osteogenic and Adipogenic Differentiation of Human Periodontal Ligament Stem Cells
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Human periodontal ligament stem cells were seeded at a density of 1 × 105 cells/well in 12‐well plates. For osteogenic differentiation, cells were cultured in an osteogenic induction medium (phenol red‐free α‐MEM supplemented with 10% FBS, 10 mmol/L β‐glycerophosphate, 10 nmol/L dexamethasone and 50 μg/mL ascorbic acid).22 The medium was replaced every other day with fresh osteogenic induction medium. Three weeks later, cells were fixed with 4% paraformaldehyde and stained by the Alizarin red. After dissolving with 10% (w/v) cetylpyridinium chloride (Sigma‐Aldrich), quantification of stained nodules was detected by automatic microplate reader at 562 nm.
For adipogenic differentiation, cells were cultured in adipogenic medium (Cyagen Biosciences Inc, Santa Clara, CA, USA). Two weeks later, cells were fixed with 4% paraformaldehyde and stained with oil red O.
For adipogenic differentiation, cells were cultured in adipogenic medium (Cyagen Biosciences Inc, Santa Clara, CA, USA). Two weeks later, cells were fixed with 4% paraformaldehyde and stained with oil red O.
5
Multilineage Differentiation of HUC-MSCs
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The adherent HUC-MSCs were passaged and assayed at passage 3 (P3). The P3 cells were added with osteogenic medium (Cyagen Biosciences Inc., Guangzhou, China) and chondrogenic medium (Cyagen Biosciences Inc.) for 21-day cell culture and with adipogenic medium (Cyagen Biosciences Inc.) for 14-day cell culture. After the cell culture, the osteogenic, chondrogenic, and adipogenic differentiation abilities of the HUC-MSCs were respectively identified by alizarin red staining (Shanghai Yuanye Biotechnology Co, Ltd., Shanghai, China), Alcian blue staining (Shanghai Yuanye Biotechnology Co, Ltd.), and oil red O staining (Shanghai Yuanye Biotechnology Co, Ltd.), and the cells were then observed under a microscope and photographed. Flow cytometry was used to examine the expression of CD34 (a negative marker for stem cells), CD29, CD90, and CD44 (positive markers for stem cells) in the P3 adherent cells.
6
Adipogenic Differentiation of Cells
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2 × 105 cells were seeded in 6‐well plates. When cells reached 80% confluence, adipogenic medium (Cyagen) was used to induce adipogenic differentiation and refreshed according to the manufacturer's protocol. Oil Red O staining was conducted and quantified at Day 21.
7
Osteogenic and Adipogenic Cell Culture
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Alpha-minimum essential medium was purchased from Gibco (Grand Island, NY, USA). The osteogenic medium consisted of a standard culture medium supplemented with 100 µM L-ascorbic-2-phosphate, 20 µM dexamethasone, and 2 M β-glycerophosphate purchased from Sigma-Aldrich. Adipogenic medium was purchased from Cyagen Biosciences Inc. (Sunnyvale, CA, USA). The CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) kit was obtained from Promega Corporation. Zein (Z3625), gelatin from porcine skin (gel strength = 300 g Bloom, Type A), and collagenase type I were purchased from Sigma. 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP, 99%) was purchased from Energy Chemical. Nano hydroxyapatite (nHAp, ≥97%, <100 nm particle size) was purchased from Aladdin.
8
Characterization and Differentiation of hucMSCs
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HucMSCs (7530, ScienCell Research Laboratories, CA, USA) were subcultured to passage 4 in the medium (7501, ScienCell Research Laboratories).
The typical surface markers of hucMSCs were tested by ow cytometry. HucMSCs were resuspended in bovine serum albumin and combined with CD29 (1:100, 553715, BD Biosciences, CA, USA), CD34 (1:200, ab81289, Abcam, MA, USA), CD73 (1:50, 550257, BD Biosciences) and CD90 (1:100, 555595, BD Biosciences), respectively. HucMSCs were detected by ow cytometry after resuspending with phosphatebuffered saline (PBS).
HucMSCs were placed in osteogenic medium (0.1 mM dexamethasone, 10 mM β-glycerophosphate and 50 mM ascorbyl phosphate) or adipogenic medium (Cyagen Biosciences, CA, USA). Two weeks later, the osteogenic and adipogenic differentiation potentials were evaluated by Alizarin Red and Oil Red O staining [16, 17] .
Cell transfection miR-342-3p negative control (NC), miR-342-3p agomir and miR-342-3p antagomir (Sangon, Shanghai, China) were utilized to transfect hucMSCs using Lipofectamine 3000 (Invitrogen, CA, USA) [18] .
The typical surface markers of hucMSCs were tested by ow cytometry. HucMSCs were resuspended in bovine serum albumin and combined with CD29 (1:100, 553715, BD Biosciences, CA, USA), CD34 (1:200, ab81289, Abcam, MA, USA), CD73 (1:50, 550257, BD Biosciences) and CD90 (1:100, 555595, BD Biosciences), respectively. HucMSCs were detected by ow cytometry after resuspending with phosphatebuffered saline (PBS).
HucMSCs were placed in osteogenic medium (0.1 mM dexamethasone, 10 mM β-glycerophosphate and 50 mM ascorbyl phosphate) or adipogenic medium (Cyagen Biosciences, CA, USA). Two weeks later, the osteogenic and adipogenic differentiation potentials were evaluated by Alizarin Red and Oil Red O staining [16, 17] .
Cell transfection miR-342-3p negative control (NC), miR-342-3p agomir and miR-342-3p antagomir (Sangon, Shanghai, China) were utilized to transfect hucMSCs using Lipofectamine 3000 (Invitrogen, CA, USA) [18] .
9
Multilineage Differentiation of hPDLSCs
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hPDLSCs were induced in OS, adipogenic medium (Cyagen Biosciences, Santa Clara, CA, USA), and chondrogenic medium (Cyagen Biosciences) for 21 days. Next, the cells were fixed in 4% paraformaldehyde and stained with 1% Alizarin red staining (ARS) solution (Cyagen Biosciences) for the osteogenesis assay. Additionally, 3 mg/mL oil red O (Sigma-Aldrich) was used for adipogenesis analysis and Alcian blue staining for the chondrogenic assay. The images were viewed using an inverted microscope (Zeiss AG, Oberkochen, Germany).
10
Multilineage Differentiation Potential of Adherent Cells
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The third generation of the adherent cells was cultured in an osteogenic medium (Cyagen Biosciences, Guangzhou, China) for 21 days or in an adipogenic medium (Cyagen Biosciences, Guangzhou, China) for 14 days. After the cultivation, the osteogenesis‐induced cells were stained in alizarin red (Yuanye Bio‐Technology Co., Ltd., Shanghai, China) and the adipogenesis‐induced cells were stained in oil red O (Yuanye Bio‐Technology Co., Ltd., Shanghai, China). The cells were observed and photographed under a microscope. The expressions of CD34 and CD90 were measured by flow cytometry for identification of the stemness of these cells.
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