Preadipocytes and Mouse 3T3-L1 Adipocytes...Day 7 Day 14 Day 21 Day 28 Day 0 (Preadipocytes) Day 0...
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Day 7 Day 14 Day 21 Day 28 Day 0 (Preadipocytes) Day 0 Day 7 Day 14 Day 21 Day 28 A B Increasing Leptin as Detected by Array and ELISA Measurement Method Leptin Mean Pixel Density 0 10000 20000 30000 40000 50000 Leptin Concentration (pg/mL) 0 1000 2000 3000 4000 5000 Array ELISA 2 1 3 4 5 10 11 12 9 6 Day 7 Day 14 Day 21 Day 28 Day 0 2 1 3 4 5 10 11 12 9 6 2 1 3 4 5 10 11 12 9 6 2 1 3 4 5 10 11 12 9 6 7 8 Human Adipokine Secretion During Adipocyte Differentiation Mean Pixel Density Mean Pixel Density 0 10000 Adiponectin/ Acrp-30 Adiponectin/ Acrp-30 Angiopoietin- like 2 Cathepsin D Cathepsin L Complement Factor D IGFBP-2 IGFBP-2 IL-6 IL-8/ CXCL8 Leptin Leptin Adiponectin/ Acrp-30 IGFBP-2 Leptin MCP-1/ CCL2 MIF TIMP-1 1. Adiponectin/Acrp-30 2. Angiopoietin-like 2 3. Cathepsin D 4. Cathepsin L 5. Complement Factor D 6. IGFBP-2 7. IL-6 8. IL-8/CXCL8 9. Leptin 10. MCP-1/CCL2 11. MIF 12. TIMP-1 20000 30000 40000 50000 Day 0 Day 7 Day 14 Day 21 Day 28 Day 0 Day 14 Day 28 Day 0 Day 14 Day 28 B A C Array Human Analyte Profiles 0 10000 20000 30000 40000 50000 ELISA Human Analyte Concentrations Adiponectin/ACRP-30 Concentration (ng/mL) 0 100 200 300 400 500 Leptin and IGFBP-2 Concentration (pg/mL) 0 5000 10000 15000 20000 Streptavidin-HRP Detection Antibody Target Analyte Analyte Array Membrane Biotin LIGHT Abstract Adipose tissue secretes a variety of bioactive peptides, called adipokines, that act in an autocrine, paracrine, or endocrine manner to regulate energy metabolism. In vitro cultured primary adipocytes and cell lines, such as 3T3-L1 mouse embryonic fibroblast adipose-like cells, are commonly used models for studying adipocyte functions. In order to better understand adipogenesis in these model systems, we analyzed adipokine secretion during in vitro differentiation of human preadipocytes and mouse 3T3-L1 cells. 3T3-L1 cells were untreated or differentiated with methylisobutylxanthine (115 mg/mL), insulin (10 mg/mL), and dexamethasone (390 ng/mL) for 48 hours, and then with additional insulin (10 mg/mL) for the next 48 hours. The cells were then grown in standard media that was changed every 48 hours for 96 hours. Human subcutaneous preadipocytes (BMI=30, female) were differentiated into primary human adipocytes using differentiation media from ZenBio. Phase-contrast microscopy revealed the formation of visible lipid droplets during the differentiation of 3T3-L1 cells and human preadipocytes. Proteome Profiler ™ Mouse Adipokine Antibody Arrays were used to screen 3T3-L1 cell culture supernates collected on days 2, 4, and 6. Proteome Profiler ™ Human Adipokine Antibody Arrays were used to screen adipocyte cell culture supernates collected on days 0, 7, 14, 21, and 28. Array data for select adipokines were confirmed using Quantikine ® ELISAs. In conclusion, our data suggest that the combined use of antibody arrays and ELISAs is an effective method by which to study adipokine secretion during mouse and human in vitro adipocyte differentiation. Materials & Methods Cell Culture 3T3-L1 mouse embryonic fibroblast adipocyte-like cells were untreated or differentiated with methylisobutylxanthine (115 mg/mL), insulin (10 mg/mL), and dexamethasone (390 ng/mL) for 48 hours, followed by additional insulin (10 mg/mL) for the next 48 hours. The cells were then grown in standard media that was changed every 48 hours for 96 hours. Human subcutaneous preadipocytes (BMI=30, female) were differentiated into primary human adipocytes using differentiation media from ZenBio. Proteome Profiler ™ Antibody Array Analysis Human Adipokine Antibody Arrays (R&D Systems, Catalog # ARY024) were used to screen adipocyte cell culture supernates collected on days 0, 7, 14, 21, and 28. Mouse Adipokine Antibody Arrays (R&D Systems, Catalog # ARY013) were used to screen 3T3-L1 cell culture supernates collected on days 2, 4, and 6. 500 μL of cell culture supernate was run on each array. Quantikine ® ELISA Analysis Adipocyte cell culture supernates collected on days 0, 14, and 28 were analyzed using the Human Leptin ELISA Kit (R&D Systems, Catalog # DLP00), the Human IGFBP-2 ELISA Kit (R&D Systems, Catalog # DGB200), and the Human Adiponectin/Acrp-30 ELISA Kit (R&D Systems, Catalog # DRP300). Summary The Human and Mouse Proteome Profiler™ Adipokine Array Kit detected changes in adipokine secretion during the differentiation of adipocytes and adipocyte-like cells. • Leptin, Adiponectin/Acrp30, Lipocalin-2/NGAL, and Resistin protein levels increased during differentiation. • IL-6 and IL-8 protein levels decreased during differentiation. Changes in analyte concentration for Adiponectin/Acrp-30, Leptin, and IGFBP-2 correlated well between array and quantitative ELISA data. The combined use of Proteome Profiler ™ Antibody Arrays and Quantikine ® ELISAs is an effective method to study adipokine secretion during mouse and human in vitro adipocyte differentiation. Profiling Adipokines Secreted During Differentiation of Human Preadipocytes and Mouse 3T3-L1 Adipocytes J. Rivard, R. Weller Roska, N. Lee, A. James, K. Brumbaugh, and G. Wegner | R&D Systems, Inc., 614 McKinley Place NE, Minneapolis, MN, 55413 Trademarks and registered trademarks are the property of their respective owners. For research use or manufacturing purposes only. PS_Obesity_1990 Figure 1. Profiling Human Adipokines Secreted During Adipocyte Differentiation Figure 2. Leptin Concentrations Correlate with Increasing Lipid Droplets Figure 3. Profiling Mouse Adipokines Secreted During 3T3-L1 Differentiation Proteome Profiler ™ Array Assay Principle Day 2 Day 4 Day 6 1. Adiponectin/Acrp-30 2. IGFBP-6 3. Lipoclain-2/NGAL 4. MCP-1/CCL2 5. M-CSF 6. Pentraxin-3/TSG-14 7. Pref-1 8. Resistin 9. VEGF Adiponectin/ Acrp-30 IGFBP-6 Lipoclain-2/ NGAL MCP-1/CCL2 M-CSF Pentraxin-3/ TSG-14 Pref-1 Resistin VEGF A B Mouse 3T3-L1 Adipokine Secretion During Differentiation Mean Pixel Density 0 10000 20000 30000 40000 50000 60000 Day 2 Day 4 Day 6 2 1 3 5 6 4 7 8 9 2 5 6 4 7 9 2 1 3 5 6 4 7 9 Cell culture supernates from human undifferentiated preadipocytes and adipocytes were analyzed semi- quantitatively using the Human Adipokine Array Kit and quantitatively using ELISA kits. (A) Arrays incubated with supernates collected on days 0, 7, 14, 21, and 28 of differentiation are shown (2 minute X-ray film exposure). (B) Histogram profiles of mean spot pixel density for select analytes are shown. (C) The mean pixel densities (left) for Adiponectin/Acrp-30, Leptin, and IGFBP-2 on days 0, 14, and 28 are compared to the concentrations (right) measured by ELISA kits. The Leptin concentration in cell culture supernates from human undifferentiated preadipocytes (Day 0) and adipocytes on days 7, 14, 21, and 28 of differentiation was measured semi-quantitatively using the Human Adipokine Array Kit and quantitatively using the ELISA kit. (A) Phase-contrast microscopy images of the human preadipocytes (Day 0) and human adipocytes (Days 7, 14, 21, and 28) that were used to obtain the cell culture supernates from Figure 1. Lipid droplets increase in number and size over the 28 day culture period. (B) Leptin concentrations measured by array and ELISA increase over the 28 day culture period. Cell culture supernates from 3T3-L1 mouse embryonic fibroblast adipocyte-like cells were analyzed using the Mouse Adipokine Array kit. (A) Arrays incubated with supernates collected on days 2, 4, and 6 of differentiation are shown (5 minute X-ray film exposure). (B) Histogram profiles of mean spot pixel density for select analytes are shown.
Transcript of Preadipocytes and Mouse 3T3-L1 Adipocytes...Day 7 Day 14 Day 21 Day 28 Day 0 (Preadipocytes) Day 0...
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1. Adiponectin/Acrp-302. Angiopoietin-like 23. Cathepsin D4. Cathepsin L5. Complement Factor D6. IGFBP-2
7. IL-68. IL-8/CXCL89. Leptin10. MCP-1/CCL211. MIF12. TIMP-1
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LIGHT
AbstractAdipose tissue secretes a variety of bioactive peptides, called adipokines, that act in an autocrine, paracrine, or endocrine manner to regulate energy metabolism. In vitro cultured primary adipocytes and cell lines, such as 3T3-L1 mouse embryonic fi broblast adipose-like cells, are commonly used models for studying adipocyte functions. In order to better understand adipogenesis in these model systems, we analyzed adipokine secretion during in vitro differentiation of human preadipocytes and mouse 3T3-L1 cells. 3T3-L1 cells were untreated or differentiated with methylisobutylxanthine (115 mg/mL), insulin (10 mg/mL), and dexamethasone (390 ng/mL) for 48 hours, and then with additional insulin (10 mg/mL) for the next 48 hours. The cells were then grown in standard media that was changed every 48 hours for 96 hours. Human subcutaneous preadipocytes (BMI=30, female) were differentiated into primary human adipocytes using differentiation media from ZenBio. Phase-contrast microscopy revealed the formation of visible lipid droplets during the differentiation of 3T3-L1 cells and human preadipocytes. Proteome Profi ler™ Mouse Adipokine Antibody Arrays were used to screen 3T3-L1 cell culture supernates collected on days 2, 4, and 6. Proteome Profi ler™ Human Adipokine Antibody Arrays were used to screen adipocyte cell culture supernates collected on days 0, 7, 14, 21, and 28. Array data for select adipokines were confi rmed using Quantikine® ELISAs. In conclusion, our data suggest that the combined use of antibody arrays and ELISAs is an effective method by which to study adipokine secretion during mouse and human in vitro adipocyte differentiation.
Materials & MethodsCell Culture3T3-L1 mouse embryonic fi broblast adipocyte-like cells were untreated or differentiated with methylisobutylxanthine (115 mg/mL), insulin (10 mg/mL), and dexamethasone (390 ng/mL) for 48 hours, followed by additional insulin (10 mg/mL) for the next 48 hours. The cells were then grown in standard media that was changed every 48 hours for 96 hours. Human subcutaneous preadipocytes (BMI=30, female) were differentiated into primary human adipocytes using differentiation media from ZenBio.
Proteome Profi ler™ Antibody Array AnalysisHuman Adipokine Antibody Arrays (R&D Systems, Catalog # ARY024) were used to screen adipocyte cell culture supernates collected on days 0, 7, 14, 21, and 28. Mouse Adipokine Antibody Arrays (R&D Systems, Catalog # ARY013) were used to screen 3T3-L1 cell culture supernates collected on days 2, 4, and 6. 500 μL of cell culture supernate was run on each array.
Quantikine® ELISA AnalysisAdipocyte cell culture supernates collected on days 0, 14, and 28 were analyzed using the Human Leptin ELISA Kit (R&D Systems, Catalog # DLP00), the Human IGFBP-2 ELISA Kit (R&D Systems, Catalog # DGB200), and the Human Adiponectin/Acrp-30 ELISA Kit (R&D Systems, Catalog # DRP300).
SummaryThe Human and Mouse Proteome Profi ler™ Adipokine Array Kit detected changes in adipokine secretion during the differentiation of adipocytes and adipocyte-like cells.
• Leptin, Adiponectin/Acrp30, Lipocalin-2/NGAL, and Resistin protein levels increased during differentiation.
• IL-6 and IL-8 protein levels decreased during differentiation.
Changes in analyte concentration for Adiponectin/Acrp-30, Leptin, and IGFBP-2 correlated well between array and quantitative ELISA data.
The combined use of Proteome Profi ler™ Antibody Arrays and Quantikine® ELISAs is an effective method to study adipokine secretion during mouse and human in vitro adipocyte differentiation.
Profi ling Adipokines Secreted During Differentiation of Human Preadipocytes and Mouse 3T3-L1 Adipocytes
J. Rivard, R. Weller Roska, N. Lee, A. James, K. Brumbaugh, and G. Wegner | R&D Systems, Inc., 614 McKinley Place NE, Minneapolis, MN, 55413
RnDSy-lu-2945
Trademarks and registered trademarks are the property of their respective owners. For research use or manufacturing purposes only. PS_Obesity_1990
Figure 1. Profi ling Human Adipokines Secreted During Adipocyte Differentiation Figure 2. Leptin Concentrations Correlate with Increasing Lipid Droplets
Figure 3. Profi ling Mouse Adipokines Secreted During 3T3-L1 Differentiation
Proteome Profi ler™ Array Assay PrincipleDay 2
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1. Adiponectin/Acrp-302. IGFBP-63. Lipoclain-2/NGAL4. MCP-1/CCL25. M-CSF
6. Pentraxin-3/TSG-147. Pref-18. Resistin9. VEGF
Adiponectin/Acrp-30
IGFBP-6 Lipoclain-2/NGAL
MCP-1/CCL2 M-CSF Pentraxin-3/TSG-14
Pref-1 Resistin VEGF
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Cell culture supernates from human undifferentiated preadipocytes and adipocytes were analyzed semi-quantitatively using the Human Adipokine Array Kit and quantitatively using ELISA kits. (A) Arrays incubated with supernates collected on days 0, 7, 14, 21, and 28 of differentiation are shown (2 minute X-ray fi lm exposure). (B) Histogram profi les of mean spot pixel density for select analytes are shown. (C) The mean pixel densities (left) for Adiponectin/Acrp-30, Leptin, and IGFBP-2 on days 0, 14, and 28 are compared to the concentrations (right) measured by ELISA kits.
The Leptin concentration in cell culture supernates from human undifferentiated preadipocytes (Day 0) and adipocytes on days 7, 14, 21, and 28 of differentiation was measured semi-quantitatively using the Human Adipokine Array Kit and quantitatively using the ELISA kit. (A) Phase-contrast microscopy images of the human preadipocytes (Day 0) and human adipocytes (Days 7, 14, 21, and 28) that were used to obtain the cell culture supernates from Figure 1. Lipid droplets increase in number and size over the 28 day culture period. (B) Leptin concentrations measured by array and ELISA increase over the 28 day culture period.
Cell culture supernates from 3T3-L1 mouse embryonic fi broblast adipocyte-like cells were analyzed using the Mouse Adipokine Array kit. (A) Arrays incubated with supernates collected on days 2, 4, and 6 of differentiation are shown (5 minute X-ray fi lm exposure). (B) Histogram profi les of mean spot pixel density for select analytes are shown.
