Oil Red O staining was performed by using a Lipid assay kit (Cosmo Bio, Sapporo, Japan) in accordance with the manufacturer's instructions. Briefly, differentiated 3T3-L1 cells were washed in Dulbecco's phosphate-buffered saline (D-PBS), fixed in 10% formalin neutral buffer solution (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) for 1 h, and stained with Oil Red O for 1 h. Oil Red O-stained cells were observed by using a microscope (IX70; Olympus, Tokyo, Japan). Six stained areas per well were then measured by using Win ROOF® ver. 5.8 (MITANI Corporation, Tokyo, Japan). Cell areas that were stained with LAT-A, PGF2α, or OMD were expressed as percentages of those in untreated control cultures.
Lipid assay kit
Manufactured by Cosmo Bio
Sourced in Japan
The Lipid Assay Kit is a laboratory tool designed to quantify lipid concentrations in various samples. It provides a straightforward and reliable method for the colorimetric determination of lipid levels.
Automatically generated - may contain errors
Lab products found in correlation
Calcified nodule staining kit, by Cosmo Bio (3 mentions)
Formalin neutral buffer solution, by Fujifilm (2 mentions)
Oricell mesenchymal stem cell osteogenic differentiation medium, by Cyagen (2 mentions)
Mesenchymal stem cell adipogenesis kit, by Merck Group (1 mentions)
Mesenchymal stem cell osteogenesis kit, by Merck Group (1 mentions)
Stemxvivo adipogenic supplement, by R&D Systems (1 mentions)
Stemxvivo mouse rat osteogenic supplement, by R&D Systems (1 mentions)
Stemxvivo osteogenic adipogenic base media, by Cosmo Bio (1 mentions)
Oricell mesenchymal stem cell adipogenic differentiation medium, by Cyagen (1 mentions)
Bz x700 all in one fluorescence microscope, by Keyence (1 mentions)
Multiskan fc microplate photometer, by Thermo Fisher Scientific (1 mentions)
Eclipse ti, by Nikon (1 mentions)
Infinit m200, by Tecan (1 mentions)
Total carbohydrate colorimetric assay kit, by Abcam (1 mentions)
Neutral buffered formalin, by Fujifilm (1 mentions)
Sunrise remote, by Tecan (1 mentions)
9 protocols using lipid assay kit
1
Oil Red O Staining of Adipocytes
2
Adipocyte and Osteocyte Differentiation of BM-MSCs
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To induce adipocyte or osteocyte differentiation, we incubated culture-expanded BM-MSCs at passage 3 with either adipogenic medium or osteogenic medium using a Mesenchymal Stem Cell Adipogenesis Kit or Mesenchymal Stem Cell Osteogenesis Kit (Chemicon International, Temecula, CA). Three or four days later, intracellular lipid droplets were stained with a Lipid Assay kit (Cosmo Bio, Tokyo) according to the manufacturer’s instructions. Alkaline phosphatase activity was visualised using an Alkaline Phosphatase (ALP) staining kit (Primary Cell Co., Hokkaido, Japan) according to the manufacturer’s instructions.
3
Adipogenic and Osteogenic Differentiation Assays
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Adipogenic differentiation was examined using OriCell™ Mesenchymal Stem Cell Adipogenic Differentiation Medium (GUXMX-90031, Cyagen Biosciences) and the Lipid Assay Kit (AK09F, Cosmo Bio Co. Ltd, Tokyo, Japan) according to the manufacturer's instructions. Osteogenic differentiation was examined using OriCell™ Mesenchymal Stem Cell Osteogenic Differentiation Medium (GUXMX-90021, Cyagen Biosciences) and the Calcified Nodule Staining Kit (AK21, Cosmo Bio Co. Ltd, Tokyo, Japan) according to the manufacturer's instructions.
4
Adipogenic and Osteogenic Differentiation Protocols
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Adipogenic differentiation was performed using StemXVivo Adipogenic Supplement (CCM011; R&D Systems, Minneapolis, MN, USA), StemXVivo Osteogenic/Adipogenic Base Media, and a Lipid Assay Kit (AK09F; Cosmo Bio Co., Ltd., Tokyo, Japan) according to the manufacturer’s instructions. Osteogenic differentiation was performed using StemXVivo Mouse/Rat Osteogenic Supplement (CCM009; R&D Systems), StemXVivo Osteogenic/Adipogenic Base Media and a Calcified Nodule Staining Kit (AK21, Cosmo Bio Co., Ltd.) according to the manufacturer’s instructions.
5
Mesenchymal Stem Cell Adipogenic and Osteogenic Differentiation
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Adipogenic differentiation was performed using OriCellTM Mesenchymal Stem Cell Adipogenic Differentiation Medium (GUXMX-90031; Cyagen Biosciences, Santa Clara, CA, USA) and a Lipid Assay Kit (AK09F; Cosmo Bio Co., Ltd., Tokyo, Japan) according to the manufacturer’s instructions. Osteogenic differentiation was performed using OriCell™ Mesenchymal Stem Cell Osteogenic Differentiation Medium (GUXMX-90021, Cyagen Biosciences) and a Calcified Nodule Staining Kit (AK21, Cosmo Bio Co., Ltd.) according to the manufacturer’s instructions.
6
Measuring Intracellular Triglycerides in 3T3-L1 Adipocytes
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At 24 hr after treatment, intracellular TG levels in 3T3-L1 adipocytes were determined employing a commercially available Lipid Assay kit (Cosmo Bio, Tokyo, Japan) [15 (link)], according to the manufacturer's protocol, and the values obtained were adjusted to the intracellular total protein contents. For Lipid Assay kit, cells were fixed overnight at room temperature with 10% formalin neutral buffer solution (Wako) and then stained with oil red O solution for 15 min. Oil red O solution was then removed, and the cells were washed. After drying, the cells were observed with a KEYENCE BZ-X700 All-in-one Fluorescence Microscope and extraction reagent was added to measure dye extraction (540 nm) with a plate reader, to allow calculation of the TG contents.
7
Quantitative Lipid Accumulation Analysis
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Lipid accumulation in 3T3-L1 cells or preadipocytes isolated from mice was detected by Oil Red O staining utilizing Lipid Assay Kit (Cosmo Bio, Hokkaido, Japan). Cells were rinsed with phosphate-buffered saline and fixed in 10% Formaldehyde Solution (Wako Pure Chemical Industries) overnight. The fixed cells were washed with purified water and stained with Oil Red O solution for 15 min. After three washes, cells in purified water were imaged with ECLIPSE Ti (Nikon). For the quantification, isopropanol was applied to the stained cells, and absorbance at 540 nm of the extracted solution was measured with the Multiskan FC Microplate Photometer (Thermo Scientific, Illinois, USA).
8
Lipid and Carbohydrate Analysis in Cells
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Observation and measurements of lipids in cells were performed by Oil Red O staining using a Lipid Assay kit (Cosmo Bio, Tokyo, Japan). Carbohydrates were observed by PAS staining24 (link) and their levels were measured using the phenol-sulfuric acid method with a Total Carbohydrate Colorimetric Assay kit (BioVision, Milpitas, CA). Colorimetry of lipids and carbohydrates was carried out at 540 and 490 nm, respectively, using an InfinitM200 (TECAN, Mannendorf, Switzerland).
9
Adipocyte Differentiation Quantification via ORO Staining
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Oil Red O (ORO) staining was performed on the Control group, as well as the ND and DI groups on days 5 and 10, to determine the progression of adipocyte differentiation, which was shown by lipid droplet accumulation. Cells were washed with PBS and xed with 10% neutral-buffered formalin (pH 7.4; FUJIFILM Wako Pure Chemical Corporation) overnight at room temperature. Further, cells were stained with a freshly prepared ORO staining solution (Lipid Assay Kit; Cosmo Bio, Tokyo, Japan) according to the manufacturer's instructions. Brie y, after staining for at least 15 minutes at room temperature, ORO staining solution was removed, and the stained cells were washed with distilled water (dH 2 O) three times.
Images of the cells were captured using an inverted light microscope (Olympus IX71; Olympus, Tokyo, Japan). Isopropanol was used to extract the dye from the stained lipid droplets. Semi-quantitative approach by measuring the absorbance of the dye extraction using a microplate reader (Tecan Sunrise Remote, Grödig, Austria) at 492 nm was performed to quantify the amount of oil droplets.
Images of the cells were captured using an inverted light microscope (Olympus IX71; Olympus, Tokyo, Japan). Isopropanol was used to extract the dye from the stained lipid droplets. Semi-quantitative approach by measuring the absorbance of the dye extraction using a microplate reader (Tecan Sunrise Remote, Grödig, Austria) at 492 nm was performed to quantify the amount of oil droplets.
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