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Flow cytometric analysis of C-Peptide expression in Human Insulin-transfected 293F cells and a Mouse insulinoma cell line. Left Plot - Human 293F untransfected (dashed line histogram) and Insulin-transfected (solid line histogram) cells were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655) and permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050). The cells were then washed and stained with Alexa Fluor™ 488 Mouse Anti-C-Peptide antibody (Cat. No. 570993/571064; solid line histogram) at 0.5 µg/test. Right Plot - Cells from the Mouse Beta-TC-6 (Insulinoma, ATCC© CRL-3605™) cell line were fixed with BD Cytofix™ Fixation Buffer and permeabilized with BD Phosflow™ Perm Buffer III. The cells were washed and stained with either Alexa Fluor™ 488 Mouse IgG1, κ Isotype Control (Cat. No. 565572; dashed line histogram) or Alexa Fluor™ 488 Mouse Anti-C-Peptide antibody (solid line histogram) at 0.5 µg/test. The fluorescence histograms showing C-Peptide expression were derived from gated events with the forward and side light-scatter characteristics of intact cells. Flow cytometry and data analysis were performed using a BD FACSCanto™ II Flow Cytometry System and FlowJo™ Software. Data shown on this Technical Data Sheet is not lot specific.
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BD Pharmingen™ Alexa Fluor™ 488 Mouse Anti-C-Peptide
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BD® CompBeads can be used as surrogates to assess fluorescence spillover (compensation). When fluorochrome conjugated antibodies are bound to BD® CompBeads, they have spectral properties very similar to cells. However, for some fluorochromes there can be small differences in spectral emissions compared to cells, resulting in spillover values that differ when compared to biological controls. It is strongly recommended that when using a reagent for the first time, users compare the spillover on cells and BD® CompBeads to ensure that BD® CompBeads are appropriate for your specific cellular application.
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- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- Alexa Fluor® 488 fluorochrome emission is collected at the same instrument settings as for fluorescein isothiocyanate (FITC).
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The U8-424 monoclonal antibody specifically binds to human, mouse, and rat C-Peptide, the connecting peptide that links the A- and B-chains in the proinsulin molecule. The A- and B- chains and C-Peptide are encoded by the transcript of the INS gene and are produced by the β cells in the islets of Langerhans of the pancreas. As the biosynthesis of insulin proceeds, the C-Peptide is cleaved from proinsulin to form the mature insulin hormone, which is composed of the A- and B-chains linked by 2 disulfide bonds. Mature insulin and C-Peptide are stored in granules in the β cells and are released to the blood in response to metabolic signals such as glucose, the amino acids arginine and leucine, and acetylcholine. As a result, C-Peptide is released into the blood stream in an equimolar amount to insulin; the serum level of C-Peptide correlates with pancreatic β cell function and the amount of insulin being produced. The expression of C-Peptide can be used to monitor the pancreatic differentiation of pluripotent stem cells. Insulin is an evolutionarily conserved peptide hormone that binds to receptors on target cells (primarily adipose and muscle) to promote the absorption of glucose from the blood, thus regulating fat and carbohydrate metabolism. C-Peptide itself binds to many cell types, independently of the insulin receptor, and initiates several intracellular signaling cascades.
Development References (7)
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D'Amour KA, Bang AG, Eliazer S, et al . Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nat Biotechnol. 2006; 24(12):1481-1483. (Biology). View Reference
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Kelly OG, Chan MY, Martinson LA, et al. Cell-surface markers for the isolation of pancreatic cell types derived from human embryonic stem cells. Nat Biotechnol. 2011; 29(8):750-756. (Biology). View Reference
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Ko AS, Smyth DG, Marktussen J, Sundby F. The amino acid sequence of the C-peptide of human proinsulin.. Eur J Biochem. 1971; 20(2):190-9. (Biology). View Reference
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Pagliuca FW, Millman JR, Gürtler M, et al. Generation of functional human pancreatic β cells in vitro. Cell. 2014; 159(2):428-439. (Biology). View Reference
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Rezania A, Bruin JE, Riedel MJ et al. Maturation of human embryonic stem cell-derived pancreatic progenitors into functional islets capable of treating pre-existing diabetes in mice. Diabetes. 2012; 61(8):2016-2029. (Biology). View Reference
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Suckale J, Solimena M. The insulin secretory granule as a signaling hub.. Trends Endocrinol Metab. 2010; 21(10):599-609. (Biology). View Reference
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Yosten GL, Kolar GR. The Physiology of Proinsulin C-Peptide: Unanswered Questions and a Proposed Model.. Physiology (Bethesda). 2015; 30(4):327-32. (Biology). View Reference
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