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The HUVAS culture model facilitates the differentiation of unilocular human white adipocytes in vitro. A: Schematic of 2D, scaffold-free 3D, and scaffolded HUVAS cultures, done side by side using stromal vascular fraction cells from same donor and passage, cultured in the same media and with the same differentiation time, varying only the cell environment. B: Representative confocal images of BODIPY, CellMask, and DAPI-stained adipocytes on day (d)30. Scale bars for all images in Figure 1: 500 μm (main), 50 μm (inset). C–F: Quantification of lipid droplet diameter (C), total lipid droplet area (D), adipocyte unilocularity (E), and total adipocyte area (F) across isogenic 2D, 3D, and HUVAS cultures from n = 50 cells per spheroid and 3 spheroids per individual. Similar results were obtained from cells from three different cell donors. G, H: Capillary Western blot analysis on d30 of <t>PLIN1,</t> CIDEC, UCP1, PPARγ, FAS and ATGL protein and quantification normalized to total loaded protein (UCP1 blot shown here, all original blots shown in A,) from 3 technical replicates of the same individual. All total protein measurements (n = 3 per sample x 6 proteins of interest, n tot = 18) are shown in H (right panel). I: Representative confocal images of BODIPY, CellMask, and DAPI-stained 3D and HUVAS spheroids on d10, d20, d25 and d30. J, K: Quantification of lipid droplet diameter (J) and adipocyte unilocularity (K) across timepoints from n = 50 cells from one spheroid per condition, repeated at least two times. L: Relative mRNA expression levels of preadipocyte marker PDGFRA and adipogenesis marker CEBPA at d5, d10, d15, d20, d25 and d30 in HUVAS (left) and 3D (right) assessed by qPCR from 3 spheroids of the same individual and repeated twice with similar results. M: Relative mRNA expression of PPARG across timepoints in 3D compared to HUVAS analyzed as in L. N, O: Capillary Western blot for PPARγ and TOMM20 across timepoints, with quantification shown as raw chemiluminescence values by loading equal amounts of spheroid lysate, instead of equal protein amount, for each timepoint from 3 spheroids (technical replicates) of the same individual. P: mRNA expression levels of CIDEC and PLIN1 at d5, d10, d15, d20, d25 and d30 assessed by qPCR from >3 technical replicates of two different individuals. Mean and standard deviation (SD) shown in all applicable graphs throughout the paper unless specifically indicated otherwise.
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The HUVAS culture model facilitates the differentiation of unilocular human white adipocytes in vitro. A: Schematic of 2D, scaffold-free 3D, and scaffolded HUVAS cultures, done side by side using stromal vascular fraction cells from same donor and passage, cultured in the same media and with the same differentiation time, varying only the cell environment. B: Representative confocal images of BODIPY, CellMask, and DAPI-stained adipocytes on day (d)30. Scale bars for all images in Figure 1: 500 μm (main), 50 μm (inset). C–F: Quantification of lipid droplet diameter (C), total lipid droplet area (D), adipocyte unilocularity (E), and total adipocyte area (F) across isogenic 2D, 3D, and HUVAS cultures from n = 50 cells per spheroid and 3 spheroids per individual. Similar results were obtained from cells from three different cell donors. G, H: Capillary Western blot analysis on d30 of <t>PLIN1,</t> CIDEC, UCP1, PPARγ, FAS and ATGL protein and quantification normalized to total loaded protein (UCP1 blot shown here, all original blots shown in A,) from 3 technical replicates of the same individual. All total protein measurements (n = 3 per sample x 6 proteins of interest, n tot = 18) are shown in H (right panel). I: Representative confocal images of BODIPY, CellMask, and DAPI-stained 3D and HUVAS spheroids on d10, d20, d25 and d30. J, K: Quantification of lipid droplet diameter (J) and adipocyte unilocularity (K) across timepoints from n = 50 cells from one spheroid per condition, repeated at least two times. L: Relative mRNA expression levels of preadipocyte marker PDGFRA and adipogenesis marker CEBPA at d5, d10, d15, d20, d25 and d30 in HUVAS (left) and 3D (right) assessed by qPCR from 3 spheroids of the same individual and repeated twice with similar results. M: Relative mRNA expression of PPARG across timepoints in 3D compared to HUVAS analyzed as in L. N, O: Capillary Western blot for PPARγ and TOMM20 across timepoints, with quantification shown as raw chemiluminescence values by loading equal amounts of spheroid lysate, instead of equal protein amount, for each timepoint from 3 spheroids (technical replicates) of the same individual. P: mRNA expression levels of CIDEC and PLIN1 at d5, d10, d15, d20, d25 and d30 assessed by qPCR from >3 technical replicates of two different individuals. Mean and standard deviation (SD) shown in all applicable graphs throughout the paper unless specifically indicated otherwise.
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The HUVAS culture model facilitates the differentiation of unilocular human white adipocytes in vitro. A: Schematic of 2D, scaffold-free 3D, and scaffolded HUVAS cultures, done side by side using stromal vascular fraction cells from same donor and passage, cultured in the same media and with the same differentiation time, varying only the cell environment. B: Representative confocal images of BODIPY, CellMask, and DAPI-stained adipocytes on day (d)30. Scale bars for all images in Figure 1: 500 μm (main), 50 μm (inset). C–F: Quantification of lipid droplet diameter (C), total lipid droplet area (D), adipocyte unilocularity (E), and total adipocyte area (F) across isogenic 2D, 3D, and HUVAS cultures from n = 50 cells per spheroid and 3 spheroids per individual. Similar results were obtained from cells from three different cell donors. G, H: Capillary Western blot analysis on d30 of <t>PLIN1,</t> CIDEC, UCP1, PPARγ, FAS and ATGL protein and quantification normalized to total loaded protein (UCP1 blot shown here, all original blots shown in A,) from 3 technical replicates of the same individual. All total protein measurements (n = 3 per sample x 6 proteins of interest, n tot = 18) are shown in H (right panel). I: Representative confocal images of BODIPY, CellMask, and DAPI-stained 3D and HUVAS spheroids on d10, d20, d25 and d30. J, K: Quantification of lipid droplet diameter (J) and adipocyte unilocularity (K) across timepoints from n = 50 cells from one spheroid per condition, repeated at least two times. L: Relative mRNA expression levels of preadipocyte marker PDGFRA and adipogenesis marker CEBPA at d5, d10, d15, d20, d25 and d30 in HUVAS (left) and 3D (right) assessed by qPCR from 3 spheroids of the same individual and repeated twice with similar results. M: Relative mRNA expression of PPARG across timepoints in 3D compared to HUVAS analyzed as in L. N, O: Capillary Western blot for PPARγ and TOMM20 across timepoints, with quantification shown as raw chemiluminescence values by loading equal amounts of spheroid lysate, instead of equal protein amount, for each timepoint from 3 spheroids (technical replicates) of the same individual. P: mRNA expression levels of CIDEC and PLIN1 at d5, d10, d15, d20, d25 and d30 assessed by qPCR from >3 technical replicates of two different individuals. Mean and standard deviation (SD) shown in all applicable graphs throughout the paper unless specifically indicated otherwise.
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The HUVAS culture model facilitates the differentiation of unilocular human white adipocytes in vitro. A: Schematic of 2D, scaffold-free 3D, and scaffolded HUVAS cultures, done side by side using stromal vascular fraction cells from same donor and passage, cultured in the same media and with the same differentiation time, varying only the cell environment. B: Representative confocal images of BODIPY, CellMask, and DAPI-stained adipocytes on day (d)30. Scale bars for all images in Figure 1: 500 μm (main), 50 μm (inset). C–F: Quantification of lipid droplet diameter (C), total lipid droplet area (D), adipocyte unilocularity (E), and total adipocyte area (F) across isogenic 2D, 3D, and HUVAS cultures from n = 50 cells per spheroid and 3 spheroids per individual. Similar results were obtained from cells from three different cell donors. G, H: Capillary Western blot analysis on d30 of <t>PLIN1,</t> CIDEC, UCP1, PPARγ, FAS and ATGL protein and quantification normalized to total loaded protein (UCP1 blot shown here, all original blots shown in A,) from 3 technical replicates of the same individual. All total protein measurements (n = 3 per sample x 6 proteins of interest, n tot = 18) are shown in H (right panel). I: Representative confocal images of BODIPY, CellMask, and DAPI-stained 3D and HUVAS spheroids on d10, d20, d25 and d30. J, K: Quantification of lipid droplet diameter (J) and adipocyte unilocularity (K) across timepoints from n = 50 cells from one spheroid per condition, repeated at least two times. L: Relative mRNA expression levels of preadipocyte marker PDGFRA and adipogenesis marker CEBPA at d5, d10, d15, d20, d25 and d30 in HUVAS (left) and 3D (right) assessed by qPCR from 3 spheroids of the same individual and repeated twice with similar results. M: Relative mRNA expression of PPARG across timepoints in 3D compared to HUVAS analyzed as in L. N, O: Capillary Western blot for PPARγ and TOMM20 across timepoints, with quantification shown as raw chemiluminescence values by loading equal amounts of spheroid lysate, instead of equal protein amount, for each timepoint from 3 spheroids (technical replicates) of the same individual. P: mRNA expression levels of CIDEC and PLIN1 at d5, d10, d15, d20, d25 and d30 assessed by qPCR from >3 technical replicates of two different individuals. Mean and standard deviation (SD) shown in all applicable graphs throughout the paper unless specifically indicated otherwise.
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The HUVAS culture model facilitates the differentiation of unilocular human white adipocytes in vitro. A: Schematic of 2D, scaffold-free 3D, and scaffolded HUVAS cultures, done side by side using stromal vascular fraction cells from same donor and passage, cultured in the same media and with the same differentiation time, varying only the cell environment. B: Representative confocal images of BODIPY, CellMask, and DAPI-stained adipocytes on day (d)30. Scale bars for all images in Figure 1: 500 μm (main), 50 μm (inset). C–F: Quantification of lipid droplet diameter (C), total lipid droplet area (D), adipocyte unilocularity (E), and total adipocyte area (F) across isogenic 2D, 3D, and HUVAS cultures from n = 50 cells per spheroid and 3 spheroids per individual. Similar results were obtained from cells from three different cell donors. G, H: Capillary Western blot analysis on d30 of <t>PLIN1,</t> CIDEC, UCP1, PPARγ, FAS and ATGL protein and quantification normalized to total loaded protein (UCP1 blot shown here, all original blots shown in A,) from 3 technical replicates of the same individual. All total protein measurements (n = 3 per sample x 6 proteins of interest, n tot = 18) are shown in H (right panel). I: Representative confocal images of BODIPY, CellMask, and DAPI-stained 3D and HUVAS spheroids on d10, d20, d25 and d30. J, K: Quantification of lipid droplet diameter (J) and adipocyte unilocularity (K) across timepoints from n = 50 cells from one spheroid per condition, repeated at least two times. L: Relative mRNA expression levels of preadipocyte marker PDGFRA and adipogenesis marker CEBPA at d5, d10, d15, d20, d25 and d30 in HUVAS (left) and 3D (right) assessed by qPCR from 3 spheroids of the same individual and repeated twice with similar results. M: Relative mRNA expression of PPARG across timepoints in 3D compared to HUVAS analyzed as in L. N, O: Capillary Western blot for PPARγ and TOMM20 across timepoints, with quantification shown as raw chemiluminescence values by loading equal amounts of spheroid lysate, instead of equal protein amount, for each timepoint from 3 spheroids (technical replicates) of the same individual. P: mRNA expression levels of CIDEC and PLIN1 at d5, d10, d15, d20, d25 and d30 assessed by qPCR from >3 technical replicates of two different individuals. Mean and standard deviation (SD) shown in all applicable graphs throughout the paper unless specifically indicated otherwise.
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The HUVAS culture model facilitates the differentiation of unilocular human white adipocytes in vitro. A: Schematic of 2D, scaffold-free 3D, and scaffolded HUVAS cultures, done side by side using stromal vascular fraction cells from same donor and passage, cultured in the same media and with the same differentiation time, varying only the cell environment. B: Representative confocal images of BODIPY, CellMask, and DAPI-stained adipocytes on day (d)30. Scale bars for all images in Figure 1: 500 μm (main), 50 μm (inset). C–F: Quantification of lipid droplet diameter (C), total lipid droplet area (D), adipocyte unilocularity (E), and total adipocyte area (F) across isogenic 2D, 3D, and HUVAS cultures from n = 50 cells per spheroid and 3 spheroids per individual. Similar results were obtained from cells from three different cell donors. G, H: Capillary Western blot analysis on d30 of <t>PLIN1,</t> CIDEC, UCP1, PPARγ, FAS and ATGL protein and quantification normalized to total loaded protein (UCP1 blot shown here, all original blots shown in A,) from 3 technical replicates of the same individual. All total protein measurements (n = 3 per sample x 6 proteins of interest, n tot = 18) are shown in H (right panel). I: Representative confocal images of BODIPY, CellMask, and DAPI-stained 3D and HUVAS spheroids on d10, d20, d25 and d30. J, K: Quantification of lipid droplet diameter (J) and adipocyte unilocularity (K) across timepoints from n = 50 cells from one spheroid per condition, repeated at least two times. L: Relative mRNA expression levels of preadipocyte marker PDGFRA and adipogenesis marker CEBPA at d5, d10, d15, d20, d25 and d30 in HUVAS (left) and 3D (right) assessed by qPCR from 3 spheroids of the same individual and repeated twice with similar results. M: Relative mRNA expression of PPARG across timepoints in 3D compared to HUVAS analyzed as in L. N, O: Capillary Western blot for PPARγ and TOMM20 across timepoints, with quantification shown as raw chemiluminescence values by loading equal amounts of spheroid lysate, instead of equal protein amount, for each timepoint from 3 spheroids (technical replicates) of the same individual. P: mRNA expression levels of CIDEC and PLIN1 at d5, d10, d15, d20, d25 and d30 assessed by qPCR from >3 technical replicates of two different individuals. Mean and standard deviation (SD) shown in all applicable graphs throughout the paper unless specifically indicated otherwise.
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The HUVAS culture model facilitates the differentiation of unilocular human white adipocytes in vitro. A: Schematic of 2D, scaffold-free 3D, and scaffolded HUVAS cultures, done side by side using stromal vascular fraction cells from same donor and passage, cultured in the same media and with the same differentiation time, varying only the cell environment. B: Representative confocal images of BODIPY, CellMask, and DAPI-stained adipocytes on day (d)30. Scale bars for all images in Figure 1: 500 μm (main), 50 μm (inset). C–F: Quantification of lipid droplet diameter (C), total lipid droplet area (D), adipocyte unilocularity (E), and total adipocyte area (F) across isogenic 2D, 3D, and HUVAS cultures from n = 50 cells per spheroid and 3 spheroids per individual. Similar results were obtained from cells from three different cell donors. G, H: Capillary Western blot analysis on d30 of PLIN1, CIDEC, UCP1, PPARγ, FAS and ATGL protein and quantification normalized to total loaded protein (UCP1 blot shown here, all original blots shown in A,) from 3 technical replicates of the same individual. All total protein measurements (n = 3 per sample x 6 proteins of interest, n tot = 18) are shown in H (right panel). I: Representative confocal images of BODIPY, CellMask, and DAPI-stained 3D and HUVAS spheroids on d10, d20, d25 and d30. J, K: Quantification of lipid droplet diameter (J) and adipocyte unilocularity (K) across timepoints from n = 50 cells from one spheroid per condition, repeated at least two times. L: Relative mRNA expression levels of preadipocyte marker PDGFRA and adipogenesis marker CEBPA at d5, d10, d15, d20, d25 and d30 in HUVAS (left) and 3D (right) assessed by qPCR from 3 spheroids of the same individual and repeated twice with similar results. M: Relative mRNA expression of PPARG across timepoints in 3D compared to HUVAS analyzed as in L. N, O: Capillary Western blot for PPARγ and TOMM20 across timepoints, with quantification shown as raw chemiluminescence values by loading equal amounts of spheroid lysate, instead of equal protein amount, for each timepoint from 3 spheroids (technical replicates) of the same individual. P: mRNA expression levels of CIDEC and PLIN1 at d5, d10, d15, d20, d25 and d30 assessed by qPCR from >3 technical replicates of two different individuals. Mean and standard deviation (SD) shown in all applicable graphs throughout the paper unless specifically indicated otherwise.

Journal: Journal of Lipid Research

Article Title: Aerobic glycolysis drives differentiation of unilocular adipocytes

doi: 10.1016/j.jlr.2026.101023

Figure Lengend Snippet: The HUVAS culture model facilitates the differentiation of unilocular human white adipocytes in vitro. A: Schematic of 2D, scaffold-free 3D, and scaffolded HUVAS cultures, done side by side using stromal vascular fraction cells from same donor and passage, cultured in the same media and with the same differentiation time, varying only the cell environment. B: Representative confocal images of BODIPY, CellMask, and DAPI-stained adipocytes on day (d)30. Scale bars for all images in Figure 1: 500 μm (main), 50 μm (inset). C–F: Quantification of lipid droplet diameter (C), total lipid droplet area (D), adipocyte unilocularity (E), and total adipocyte area (F) across isogenic 2D, 3D, and HUVAS cultures from n = 50 cells per spheroid and 3 spheroids per individual. Similar results were obtained from cells from three different cell donors. G, H: Capillary Western blot analysis on d30 of PLIN1, CIDEC, UCP1, PPARγ, FAS and ATGL protein and quantification normalized to total loaded protein (UCP1 blot shown here, all original blots shown in A,) from 3 technical replicates of the same individual. All total protein measurements (n = 3 per sample x 6 proteins of interest, n tot = 18) are shown in H (right panel). I: Representative confocal images of BODIPY, CellMask, and DAPI-stained 3D and HUVAS spheroids on d10, d20, d25 and d30. J, K: Quantification of lipid droplet diameter (J) and adipocyte unilocularity (K) across timepoints from n = 50 cells from one spheroid per condition, repeated at least two times. L: Relative mRNA expression levels of preadipocyte marker PDGFRA and adipogenesis marker CEBPA at d5, d10, d15, d20, d25 and d30 in HUVAS (left) and 3D (right) assessed by qPCR from 3 spheroids of the same individual and repeated twice with similar results. M: Relative mRNA expression of PPARG across timepoints in 3D compared to HUVAS analyzed as in L. N, O: Capillary Western blot for PPARγ and TOMM20 across timepoints, with quantification shown as raw chemiluminescence values by loading equal amounts of spheroid lysate, instead of equal protein amount, for each timepoint from 3 spheroids (technical replicates) of the same individual. P: mRNA expression levels of CIDEC and PLIN1 at d5, d10, d15, d20, d25 and d30 assessed by qPCR from >3 technical replicates of two different individuals. Mean and standard deviation (SD) shown in all applicable graphs throughout the paper unless specifically indicated otherwise.

Article Snippet: TaqMan primers for human PDGFRA (Hs00998018_m1), CEBPA (Hs00269972_s1), PPARG (Hs01115513_m1), PLIN1 (Hs00160173_m1), CIDEC (Hs01032998_m1) and B2M (Hs00187842_m1) were used.

Techniques: In Vitro, Cell Culture, Staining, Western Blot, Expressing, Marker, Standard Deviation