Quantitative Flow Cytometry-Based Assays for Measuring Constitutive Secretion

  • David E. Gordon
  • Amber S. Shun-Shion
  • Asral W. Asnawi
  • Andrew A. PedenEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2233)


Constitutive secretion is predominantly measured by collecting the media from cells and performing plate-based assays. This approach is particularly sensitive to changes in cell number, and a significant amount of effort has to be spent to overcome this. We have developed a panel of quantitative flow cytometry-based assays and reporter cell lines that can be used to measure constitutive secretion. These assays are insensitive to changes in cell number making them very robust and well suited to functional genomic and chemical screens. Here, we outline the key steps involved in generating and using these assays for studying constitutive secretion.

Key words

Constitutive secretion Flow cytometry VSV-G GPI hGH Transport Assay Trafficking Biosynthetic transport 



We would like to thank the MRC (G120/952) and BBSRC (BB/L002841/1 and BB/S009566/1) for supporting this work. A. S. Shun-Shion was funded by the BBSRC White Rose DTP studentship (BB/J014443/1).


  1. 1.
    Rollins CT, Rivera VM, Woolfson DN, Keenan T, Hatada M, Adams SE, Andrade LJ, Yaeger D, van Schravendijk MR, Holt DA, Gilman M, Clackson T (2000) A ligand-reversible dimerization system for controlling protein-protein interactions. Proc Natl Acad Sci U S A 97:7096–7101. Scholar
  2. 2.
    Rivera VM, Wang X, Wardwell S, Courage NL, Volchuk A, Keenan T, Holt DA, Gilman M, Orci L, Cerasoli F Jr, Rothman JE, Clackson T (2000) Regulation of protein secretion through controlled aggregation in the endoplasmic reticulum. Science 287:826–830. Scholar
  3. 3.
    Gordon DE, Bond LM, Sahlender DA, Peden AA (2010) A targeted siRNA screen to identify SNAREs required for constitutive secretion in mammalian cells. Traffic 11:1191–1204. Scholar
  4. 4.
    Bond LM, Peden AA, Kendrick-Jones J, Sellers JR, Buss F (2011) Myosin VI and its binding partner optineurin are involved in secretory vesicle fusion at the plasma membrane. Mol Biol Cell 22:54–65. Scholar
  5. 5.
    Brandstaetter H, Kruppa AJ, Buss F (2014) Huntingtin is required for ER-to-Golgi transport and for secretory vesicle fusion at the plasma membrane. Dis Model Mech 7:1335–1340. Scholar
  6. 6.
    Lamb CA, Nuhlen S, Judith D, Frith D, Snijders AP, Behrends C, Tooze SA (2016) TBC1D14 regulates autophagy via the TRAPP complex and ATG9 traffic. EMBO J 35:281–301. Scholar
  7. 7.
    Miller CN, Smith EP, Cundiff JA, Knodler LA, Bailey Blackburn J, Lupashin V, Celli J (2017) A Brucella type IV effector targets the COG tethering complex to remodel host secretory traffic and promote intracellular replication. Cell Host Microbe 22(317–329):e7. Scholar
  8. 8.
    Song K, Gras C, Capin G, Gimber N, Lehmann M, Mohd S, Puchkov D, Rodiger M, Wilhelmi I, Daumke O, Schmoranzer J, Schurmann A, Krauss M (2019) A SEPT1-based scaffold is required for Golgi integrity and function. J Cell Sci 132:jcs225557. Scholar
  9. 9.
    Wendler F, Gillingham AK, Sinka R, Rosa-Ferreira C, Gordon DE, Franch-Marro X, Peden AA, Vincent JP, Munro S (2010) A genome-wide RNA interference screen identifies two novel components of the metazoan secretory pathway. EMBO J 29:304–314. Scholar
  10. 10.
    Winslow AR, Chen CW, Corrochano S, Acevedo-Arozena A, Gordon DE, Peden AA, Lichtenberg M, Menzies FM, Ravikumar B, Imarisio S, Brown S, O'Kane CJ, Rubinsztein DC (2010) Alpha-Synuclein impairs macroautophagy: implications for Parkinson's disease. J Cell Biol 190:1023–1037. Scholar
  11. 11.
    Wong M, Munro S (2014) Membrane trafficking. The specificity of vesicle traffic to the Golgi is encoded in the golgin coiled-coil proteins. Science 346:1256898. Scholar
  12. 12.
    Gordon DE, Chia J, Jayawardena K, Antrobus R, Bard F, Peden AA (2017) VAMP3/Syb and YKT6 are required for the fusion of constitutive secretory carriers with the plasma membrane. PLoS Genet 13:e1006698. Scholar
  13. 13.
    Mosier D (2004) Introduction for “safety considerations for retroviral vectors: a short review”. Appl Biosafety 9:68–75. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2021

Authors and Affiliations

  • David E. Gordon
    • 1
  • Amber S. Shun-Shion
    • 2
  • Asral W. Asnawi
    • 2
    • 3
  • Andrew A. Peden
    • 2
    Email author
  1. 1.Department of Cellular and Molecular PharmacologyUniversity of California San FranciscoSan FranciscoUSA
  2. 2.Department of Biomedical Science, Centre for Membrane Interactions and DynamicsUniversity of SheffieldSheffieldUK
  3. 3.Faculty of Medicine and Health SciencesUniversiti Sains Islam MalaysiaNegeri SembilanMalaysia

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