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Flow cytometric analysis of Chromogranin A expression in human neuroblastoma cells. SH-SY5Y neuroblastoma cells (ATCC CRL-2266) were fixed and permeabilized using BD Cytofix/Cytoperm™ Fixation/Permeablization Kit (Cat. No. 554714), washed twice with BD Perm/Wash™ Buffer (Cat. No. 554723) , then stained with either Purified Mouse Anti-Human Chromogranin A or Purified Mouse IgG1, κ Isotype Control (Cat. No.555746) monoclonal antibodies followed by PE Goat Anti-Mouse Ig (Multiple Adsorption) (Cat. No. 550589) second-step reagent. The fixed cells were resuspended in stain buffer and acquired on a BD LSR™ II Flow Cytometry System. The dotted line shows isotype control background staining, while the solid line represents Chromogranin A staining. The overlay histograms were derived from gated events with forward and side light-scatter characteristics of viable cells. Additionally, Purified Mouse Anti-Human Chromogranin A was negative on human embryonic stem (ES) cells (data not shown).

Western Blot analysis of Chromogranin A in human SH-SY5Y neuroblastoma lysate. SH-SY5Y neuroblastoma (ATCC CRL-2266) cell lysate was probed with Purified Mouse Anti-Human Chromogranin A monoclonal antibody at titrations of 4.0 to 0.007 ug/lane. Data shown in lanes 1,2,3 are two fold dilutions; 0.25ug, 0.125ug, 0.063ug. Chromogranin A is identified as a band of ~70 kDa.

Immunofluorescent staining of Chromogranin A in human neuroblastoma cells. SH-SY5Y neuroblastoma cells (ATCC CRL-2266) were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), permeabilized with BD Perm/Wash™ Buffer (Cat. No. 554723) and stained with Purified Mouse Anti-Human Chromogranin A monoclonal antibody (pseudo-colored green) at 1.2 µg/mL. The second-step reagent was Alexa Fluor® 488 goat anti-mouse Ig (Life Technologies), and counter staining was with DAPI (pseudo-colored blue). No Chromogranin A staining was observed on human embryonic stem cells (hESC) that were imaged similarly (data not shown). Also, no staining was observed when SH-SY5Y and hESC were stained with second-step reagent alone (data not shown). The image was captured on a BD Pathway™ 435 Cell Analyzer and merged using BD Attovision™ Software. BD Phosflow™ Perm Buffer III (Cat. No. 558050) or Triton™ X-100 are also suitable for permeabilization.


BD Pharmingen™ Purified Mouse Anti-Human Chromogranin A

BD Pharmingen™ Purified Mouse Anti-Human Chromogranin A

BD Pharmingen™ Purified Mouse Anti-Human Chromogranin A

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Any use of products other than the permitted use without the express written authorization of Becton, Dickinson and Company is strictly prohibited.
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- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- An isotype control should be used at the same concentration as the antibody of interest.
- Sodium azide is a reversible inhibitor of oxidative metabolism; therefore, antibody preparations containing this preservative agent must not be used in cell cultures nor injected into animals. Sodium azide may be removed by washing stained cells or plate-bound antibody or dialyzing soluble antibody in sodium azide-free buffer. Since endotoxin may also affect the results of functional studies, we recommend the NA/LE (No Azide/Low Endotoxin) antibody format, if available, for in vitro and in vivo use.
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- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
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Chromogranin A (CGA) is a member of the granin family of regulated secretory proteins that are found in secretory granules in endocrine and neuroendocrine cells and released in response to extracellular stimulation. Intracellularly, granins are important for targeting peptide hormones and neurotransmitters by their ability to aggregate in the low pH, high calcium environment of the trans-Golgi network. Extracellularly, peptides formed from proteolytic processing of granins regulate hormone secretion. CGA is a prohormone that can be cleaved into several biologically active peptides, such as pancreastatin, β-granin, vasostatin, catestatin, and parastatin. β-granin is an N-terminal fragment of CGA, while pancreastatin and catestatin are processed from the central region of CGA. Cells of the adrenal medulla, anterior pituitary, cerebral cortex as well as beta cells of the pancreas and a variety of tumor cell lines express CGA. The expression of CGA can be used to monitor the pancreatic differentiation of pluripotent stem cells.
Development References (5)
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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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Kroon E, Martinson LA, Kadoya K, Bang AG, et al. Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo. Nat Biotechnol. 2008; 26(4):443-452. (Biology). View Reference
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Loh YP, Cheng Y, Mahata SK, Corti A, Tota B. Chromogranin A and derived peptides in health and disease. J Mol Neurosci. 2012; 48(2):347-356. (Biology). View Reference
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Mouland AJ, Bevan S, White JH, Hendy GN. Human chromogranin A gene. Molecular cloning, structural analysis, and neuroendocrine cell-specific expression. J Biol Chem. 1994; 269(9):6918-6926. (Biology). View Reference
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Schulz TC, Young HY, Agulnick AD et al. A scalable system for production of functional pancreatic progenitors from human embryonic stem cells. PLoS ONE. 7(5)(Biology). View Reference
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