Sprague–Dawley rat adipose-derived stem cells (ADSCs) were obtained from Cyagen (Guangzhou, China). The cells were cultured in ADSC complete medium (Cyagen, China). The media were replaced every 2 days, and at passages 3-4, the cells were used for experiments. Osteogenic induction and adipogenic induction assays were employed for the identification of stem cells. For osteogenic differentiation, approximately 2 × 104 cells per well were seeded onto a 6-well plate. After the ADSCs reached 70% confluence, the media were replaced with osteogenic differentiation media (Cyagen, China). The media were replaced every 3 days. After a 2-week osteoinduction, the calcium nodules were stained with alizarin red S. For adipogenic differentiation assays, ADSCs were seeded at a density of 2 × 104 cells per well in a 6-well plate. Cells were incubated for 21 days in adipogenesis induction media (Cyagen, China), which was replaced every 3 days. The lipid droplets were stained with oil red O solution (Cyagen, China). Surface marker identification was performed using a Rat ADSC Analysis Kit (Cyagen, China) according to the manufacturer's recommended protocol.
Oil red o solution
Manufactured by Cyagen
Sourced in China
Oil Red O solution is a dye commonly used in histological staining procedures. It is designed to specifically stain lipids or fat deposits in cells and tissues. The solution contains Oil Red O dye dissolved in a suitable solvent, allowing for the effective identification and visualization of lipid-rich structures.
Automatically generated - may contain errors
Lab products found in correlation
Microplate spectrophotometer, by Agilent Technologies (3 mentions)
Cetylpyridinium chloride cpc, by Merck Group (3 mentions)
Alkaline phosphatase stain kit, by Yeasen (3 mentions)
Alizarin red s staining, by Cyagen (3 mentions)
Dexamethasone (dex), by Merck Group (3 mentions)
β glycerol phosphate, by Merck Group (3 mentions)
Ascorbic acid, by Solarbio (3 mentions)
Adipogenic induction medium, by Cyagen (2 mentions)
Adipogenic differentiation medium, by Cyagen (2 mentions)
Alizarin red s, by Cyagen (2 mentions)
Osteogenic differentiation media, by Cyagen (1 mentions)
Adipogenic differentiation media, by Cyagen (1 mentions)
Toluidine blue, by Cyagen (1 mentions)
Glutamine, by Cyagen (1 mentions)
Dexamethasone (dex), by Cyagen (1 mentions)
Insulin, by Cyagen (1 mentions)
Alizarin red solution, by Cyagen (1 mentions)
β glycerophosphate, by Cyagen (1 mentions)
Rosiglitazone, by Cyagen (1 mentions)
Bright field microscope, by Olympus (1 mentions)
13 protocols using oil red o solution
1
Adipose-Derived Stem Cell Characterization
2
Oil Red O Staining of Adipogenic DPSC
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For Oil Red O staining, DPSCs (2×105 cells/well) were first seeded in 6-well plates with growth medium until 100% confluence. The cells were then moved to a human DPSC adipogenic differentiation basal medium (Cyagen, China) containing glutamine, insulin, IBMX, rosiglitazone, and dexamethasone. This medium was changed at a schedule according to the manufacturer’s instructions. After 3∼4 cycles of induction/maintenance, the wells were washed with PBS, fixed in 4% paraformaldehyde, and stained with Oil Red O solution (Cyagen).
3
Multilineage Differentiation of Stem Cells
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Cells were seeded onto 24-well plates at a density of 2 × 104 cells/well. For the test group, cells were cultured with osteogenic, chondrogenic and adipogenic differentiation media (Cyagen, China) after reaching 60–70% confluence, respectively. For the control group, cells were cultured with USCs media. After induction for 21 days, cells were stained with Alizarin red S (ARS), Toluidine blue and Oil Red O solution (Cyagen, China) to evaluate the capability of osteogenic, chondrogenic and adipogenic differentiation, respectively.
4
Adipogenic and Osteogenic Differentiation of MSCs
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First, MSCs were grown in complete medium for adipogenic differentiation. When the cells reached 80% confluence, the medium was changed to adipogenic medium that consisted of rosiglitazone, 3-isobutyl-1-methylxanthin (IBMX), glutamine, dexamethasone and insulin (Cyagen Biosciences, Suzhou, China). Osteogenic differentiation was induced in MSCs by DMEM containing dexamethasone, ascorbic acids and β-glycerophosphate (Cyagen Biosciences, Suzhou, China). For Oil Red O staining, MSCs were fixed with 4% formaldehyde. After being washed with PBS, the cells were incubated with Oil Red O solution (Cyagen Biosciences Suzhou, China) for 30 min. The cells were then washed and examined under a microscope. For Alizarin Red S staining, the cells were incubated with Alizarin Red Solution (Cyagen Biosciences, Suzhou, China) for 30 min. Images were acquired by a microscope (Supplementary Figure S1B ).
5
Adipogenic Differentiation of PDLSCs
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The adipogenic differentiation potential of PDLSCs was detected using Oil Red O staining. Briefly, PDLSCs were seeded in 6-well plates at a density of 2 × 105 cells/well and cultured until 80–90% confluence. Cells were then incubated with adipogenic induction medium (Cyagen). The medium was refreshed every 2 days. After a 21-day adipogenic induction, cells were fixed with 4% paraformaldehyde, and the lipid droplets were stained with Oil Red O solution (Cyagen) and then photographed with an inverted microscope.
6
Adipogenic Differentiation of HPDLSCs
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HPDLSCs (2 × 105 cells/well) were cultured in 6-well plates containing adipogenic differentiation medium (Cyagen, Suzhou, China) for 21 days. Next, cells were fixed with 4% paraformaldehyde. Oil droplets were observed by staining with the Oil Red O solution (Cyagen, Suzhou, China).
7
Adipocyte Differentiation Quantification
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Cells after induced for 8 days, were seeded on 6-well plates (1×106/ml). After adherence, the cells were fixed in 10% paraformaldehyde for 20 min, washed in PBS, stained with oil red o solution (Cyagen Biosciences, Guangzhou, China) for 20 min, soaked in 60% isopropanol and then washed with PBS. Images were captured by bright field microscope (Olympus, Tokyo, Japan) and analyzed by software Image J (National Institute of Health, Maryland, USA).
8
Adipogenic Differentiation of Mouse MSCs
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NBM-MSCs and DBM-MSCs were seeded in 6-well plates at a density of 2×104 cells/cm2. Once the cells reached 90–100% confluence, ICR mouse MSC adipogenic differentiation medium [Cyagen Biosciences (Guangzhou), Inc., Guangzhou, China] was added, and the induction process was conducted according to the manufacturer's protocol. Cells were then stained with Oil Red O solution [Cyagen Biosciences (Guangzhou), Inc.) and were observed under an inverted fluorescence microscope (ECLIPSE TE2000-S; Nikon Corporation, Tokyo, Japan).
9
Adipogenic Differentiation of hMSCs
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hMSCs were seeded in 6 well plate or 24 well plate at 5 × 104 cells/cm2. When the cells reached 100% confluence, the growth medium was replaced with an induction medium for 3 days and then a maintenance medium for an additional day. The cycle of induction and maintenance was repeated at least three times for optimal induction. After the cells differentiated, they were fixed with 4% paraformaldehyde for 10 min and stained with oil red O solution (Cyagen) for over 2 h. The lipid droplets were observed and photographed by an inverted microscope (Leica, Hessen, Germany).
10
Bone and Adipogenic Differentiation of BMSCs
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For osteogenic differentiation, the BMSCs were plated in 6-well plates and cultured in osteogenic induction medium containing 10 mM β-glycerol phosphate (Sigma, USA), and 50 μM ascorbic acid (Solarbio, Beijing, China) 10−7 M dexamethasone (Sigma, USA). After 7 days’ induction, alkaline phosphatase (ALP) expressions were detected by Alkaline Phosphatase Stain kit (Yeasen, Shanghai, China) according to the manufacturer’s instructions. Images were taken by Leica Microsystems and ImageJ was applied to quantify the stained areas. After 14-days’ osteogenic induction, the calcium deposit was evaluated by Alizarin Red S staining (Cyagen Biosciences, Guangzhou, China). Images were taken by Leica Microsystems, followed by quantification using 10% cetylpyridinium chloride (CPC) (Sigma, USA) to dissolve the stained calcium deposit. The absorbance value of the dissolved solution at 562 nm was measured by microplate spectrophotometer (Bio-Tek, UK).
Adipogenic induction medium was provided by Cyagen Biosciences, Inc. (Guangzhou, China). After 12-days’ induction according to manufacture instruction, the cultured cells were stained with oil red O solution (Cyagen, Guangzhou, China), and were observed under light microscope (Leica Microsystems).
Adipogenic induction medium was provided by Cyagen Biosciences, Inc. (Guangzhou, China). After 12-days’ induction according to manufacture instruction, the cultured cells were stained with oil red O solution (Cyagen, Guangzhou, China), and were observed under light microscope (Leica Microsystems).
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