The Oil red O assay was conducted on differentiated adipocytes on day 10 in 96-well plate using adipogenesis assay kit from Cayman Chemicals (Ann Arbor, MI, US). The medium was removed from the wells and cells were fixed with 75 μL lipid droplet assay fixative. After 15 min incubation, the wells were washed five times with 100 μL wash solution. The Oil red O working solution (75 μL) was added to the wells and incubated for 20 min. The Oil red O solution was removed at the end of incubation, and the wells were washed with wash solution until no visible pink color in the wash. The pictures were taken under a microscope. The dye was extracted with 100 μL dye extraction solution. The absorbance was read at 490 nm using a FLUOstar OPTIMA plate reader (BMG Labtech, Durham, NC, USA).
Adipogenesis assay kit
Manufactured by Cayman Chemical
Sourced in United States
The Adipogenesis Assay Kit is a laboratory tool designed to measure the differentiation of preadipocytes into mature adipocytes. It provides a quantitative assessment of adipogenesis, a fundamental cellular process involved in fat tissue development.
Automatically generated - may contain errors
Lab products found in correlation
Adipogenesis differentiation medium, by Thermo Fisher Scientific (3 mentions)
Fluostar optima plate reader, by BMG Labtech (1 mentions)
Chondrogenesis differentiation medium, by Thermo Fisher Scientific (1 mentions)
Osteogenesis differentiation medium, by Thermo Fisher Scientific (1 mentions)
Alizarin red staining, by Thermo Fisher Scientific (1 mentions)
Alcian blue, by Merck Group (1 mentions)
Spectramax id3, by Molecular Devices (1 mentions)
Blasticidin, by InvivoGen (1 mentions)
Phloridzin, by Merck Group (1 mentions)
Phloretin, by Merck Group (1 mentions)
Bovine calf serum (bcs), by Merck Group (1 mentions)
Trypsin edta solution, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Chlorogenic acid, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Merck Group (1 mentions)
Caffeic acid, by Merck Group (1 mentions)
Ferulic acid, by Merck Group (1 mentions)
Penicillin streptomycin solution, by Merck Group (1 mentions)
Epicatechin, by ChromaDex (1 mentions)
Quercetin, by ChromaDex (1 mentions)
8 protocols using adipogenesis assay kit
1
Quantitative Analysis of Adipocyte Lipid Content
2
Quantitative Analysis of Stem Cell Differentiation
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For assays of adipogenesis or osteogenesis, expanded single cells during passages three to five were seeded at the density of 2.5 × 104 cells/cm2 in 24-well plates in DMEM with 10% FBS. At 90% confluence, the medium was switched to the adipogenesis differentiation medium or the osteogenesis differentiation medium, respectively (Invitrogen) and changed every 3 days. After 21 days of culturing, cells were fixed with 4% formaldehyde and stained with oil red O for adipocytes by adipogenesis Assay Kit (Cayman Chemical Company, Ann Arbor, MI, USA) or with 2% Alizarin Red for osteocytes following the manufacturer's protocol. Cells with oil droplet stained by Oil Red were quantified by measuring at OD at 492 nm in triplicate cultures. Mineralized cells with positive Alizarin Red staining (Alfa Aesar, Tewksbury, MA, USA) were quantified by measuring OD at 405 nm in triplicate cultures.
For the chondrogenesis assay, pellets were prepared by spinning down 1 × 105 cells and incubating in a 15-mL conical tube in chondrogenesis differentiation medium (Invitrogen) with the medium changed every 3 days. After 28 days of culturing, cells were fixed with 4% formaldehyde, and stained with Alcian Blue (Merck, Darmstadt, Germany).
For the chondrogenesis assay, pellets were prepared by spinning down 1 × 105 cells and incubating in a 15-mL conical tube in chondrogenesis differentiation medium (Invitrogen) with the medium changed every 3 days. After 28 days of culturing, cells were fixed with 4% formaldehyde, and stained with Alcian Blue (Merck, Darmstadt, Germany).
3
Adipogenesis Assay Using P2Y2R Inhibitor
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The Adipogenesis assay kit (Cayman Chemical) was used to study the role of P2Y2R in adipogenesis. Preadipocytes were seeded in a 96-well plate and differentiated to mature adipocytes. P2Y2R antagonist was added together during differentiation to inhibit P2Y2R activity. Seventy five microliters of Lipid Droplets Assay Fixative was added to each well and incubated for 15 min. Wells were washed with 100 μl of wash solution twice for 5 min each. Seventy five microliters of Oil-Red O working solution was added to all wells, including the background wells containing no cells, and incubated for 20 min after the wells were dried completely. Oil Red O solution was removed, and cells were washed with distilled water several times until the water contained no visible pink color. The wells were washed with 100 μl of wash solution twice for 5 min each. After the wells dried completely, 100 μl of dye extraction solution was added to each well and mixed for 30 min. The absorbance was read at 490 to 520 nm with a 96-well plate reader (SpectraMax iD3, Molecular Devices).
4
Adipogenic Differentiation Assay Protocol
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OASCs during passages three to five were seeded at a density of 2.5 × 104 cells per cm2 in 24-well PL plates in DMEM with 10% fetal bovine serum. At 90% confluence on day 3, the medium was switched to the Adipogenesis Differentiation Medium (Invitrogen, Carlsbad, CA). Torin 2 (0.02 µM), PX12 (10 µM), withaferin A (0.5 µM), isoliquiritigenin (25 µM), mitoxantrone (0.025 µM), and MLN 8054 (5 µM) were added 3 days after differentiation induction of OASC into mature adipocytes. After 21 days of culturing, the cells were fixed with 4% paraformaldehyde and stained with Oil Red O for adipocytes from the Adipogenesis Assay Kit (Cayman Chemical Company, Ann Arbor, MI) according to the manufacturer's protocol. Cells with oil droplets stained by Oil Red O were quantified via spectrophotometry at an absorbance of OD 490 nm in triplicate cultures (Fig. 1 ).
5
Establishment of SARS-CoV-2 Cell Models
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A549-hACE2 [a gift from Dr. Ben tenOever3 (link)], 293 T, HEK293, A549, and 3T3-L1 pre-adipocyte cell lines (refer to Supplementary Table 1 ) were cultured in DMEM and 10% FBS. U937, Calu-3, and HCT116 cells were cultured in RPMI, EMEM, and McCoy’s 5 A media, respectively, with 10% FBS. Caco-2 cells were cultured in EMEM with 20% FBS. Vero E6-TMPRSS2 cells were generated by lentiviral transduction of Vero E6 (CRL-1586) cells and were maintained in DMEM containing 10% FBS and 20 μg/mL blasticidin (Invivogen). All media were supplemented with glutamine and penicillin–streptomycin. EPRS1 knockdown in 3T3-L1 pre-adipocytes was generated by CRISPR, and adipocytic cell differentiation was done using Adipogenesis Assay Kit (Cayman Chemicals) per manufacturer’s protocol54 (link). HEK293‒3′-end, SPEAR, and GU-mutant cell lines were made by reporter plasmid transfection and G418 (800–1000 ng/ml) selection over five passages. A549-hACE2‒3′-end/SPEAR element and Caco-2‒3′-end/SPEAR element cell lines were made similarly with selection at 600 and 800 ng/ml of G418, respectively.
6
Quantification of Phytochemicals and Cell Assays
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The liquid chromatography standards: phloridzin, phloretin, chlorogenic acid, ferulic acid, and caffeic acid were purchased from Sigma Aldrich (Oakville, ON, Canada); catechin, epicatechin, quercetin, quercetin-3-O-galactoside and quercitin-3-O-glucoside from ChromaDex, Inc. (Santa Ana, CA, USA); quercitin-3-O-rhamnoside, quercitin-3-O-galactoside and anthocyanin standards from Indofine Chemical Company (Hillsborough, NJ, USA). The analytic solvents, chemicals, and enzymes were obtained from Sigma Aldrich (Oakville, ON, Canada). Mouse embryo 3T3-L1 cell line (ATCC, CL-173) was obtained from Cedarlane (Burlington, ON, Canada). Phenazine methosulfate (PMS) and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) were purchased from Sigma–Aldrich Canada Ltd. (Oakville, ON, Canada). Dulbecco's modified Eagle's medium (DMEM), penicillin-streptomycin solution, fetal bovine serum, bovine calf serum, trypsin-EDTA solution, dexamethasone (DEX), 3-isobutyl-1-methylxanthine (IBMX), insulin solution and all other cell biology grade chemicals were purchased from Sigma–Aldrich (Oakville, ON, Canada). Lipolysis colorimetric assay kit was purchased from Biovison Inc. (Milpitas, CA, USA) and adipogenesis assay kit from Cayman Chemical Co. (Ann Arbor, MI, USA).
7
Bimatoprost Enhances Adipocyte Differentiation
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OASCs during passages three to five were seeded at a density of 2.5 × 104 cells per cm2 in 24-well PL plates in DMEM with 10% FBS. At 90% confluence on day 3, the medium was switched to the Adipogenesis Differentiation Medium (Invitrogen, Carlsbad, CA, USA). Bimatoprost (1 μM; Selleckchem Catalog No.S1407) was added on day 6 after differentiation of OASC into mature adipocytes. After 21 days of culturing, the cells were fixed with 4% paraformaldehyde (PFA) and stained with oil red O for adipocytes from the Adipogenesis Assay Kit (Cayman Chemical Company, Ann Arbor, MI, USA) according to the manufacturer's protocol. Cells with oil droplets stained by oil red O were quantified via spectrophotometry at an absorbance of OD 492 nm in triplicate cultures.
8
Isolation and Characterization of ADSCs
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ADSCs were prepared from C57BL/6 subcutaneous fat as described previously (Gondo et al., 2008 (link)) and cultured for 2 weeks in alpha-minimum essential medium containing 20% horse serum and 1% antibiotic antimycotic (Gibco) in a 5% CO2 incubator at 37 °C. After four times passage of the culture, the adherent cells were used as ADSCs. For characterization of ADSCs, cell surface markers were analyzed by a flow cytometer using a Mouse Multipotent Mesenchymal Stromal Cell Marker Antibody Panel (R&D systems, Inc., MN). To test their capability for osteoblastic differentiation, ADSCs were cultured under a previously described condition (Gondo et al., 2004 (link)) and then stained with an anti-osteopontin antibody (R&D systems, Inc.). Induction of adipogenesis followed by Oil-Red O staining was performed using an Adipogenesis Assay Kit (Cayman Chemical Company, MI).
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