Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Volodymyr Petrenko; Camille Saini; Laurent Perrin; Charna Dibner

doi:10.3791/54673

Journal

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Genetics

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Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

JoVE Journal
Genetics

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JoVE Journal Genetics

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

8,182 Views

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06:53 min

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November 11, 2016

DOI:

10.3791/54673-v

DOI:

10.3791/54673-v

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06:53 min

November 11, 2016

•

2 Views

Volodymyr Petrenko*¹, Camille Saini*², Laurent Perrin*¹, Charna Dibner¹

¹Department of Medical Specialties, Division of Endocrinology, Diabetes, Hypertension and Nutrition, Diabetes Center,University of Geneva Medical School, Institute of Genetics and Genomics in Geneva (iGE3), ²Population Epidemiology Unit (UEP), Community Medicine,Geneva University Hospital

Transcript

The overall goal of this procedure is the parallel assessment of circadian bioluminescence and the secretory activity of human primary endocrine cells cultured and synchronized en vitro.This method can help to investigate the temporal pattern of cell secretion and how it can be regulated by cell-autonomous circadian clocks.The main advantage of this technique is that it allows us to monitor the circadian luciferins as reporter activity and, simultaneously collect outflow medium for measuring the levels of secreted hormones and myokines.To introduce circadian bioluminescence reporters into primary cells by lentiviral transduction, first, transfer the 3.5 cm petri dishes containing human myoblasts at 30%to 50%confluency to a laminar flow cabinet.Replace the human myoblast medium with 2 mL of growth medium.Then transduce the primary cell culture by pipetting lentivirus solution into the dish to obtain a multiplicity of infection equal to three.Incubate the cells overnight in a tissue-culture incubator.Change medium the next day.To achieve en vitro synchronization, add 10 micromolar adenylyl cyclase activator in 2 mL of medium per 3.5 cm petri dish containing the previously transfected primary cells.Incubate the cells for 60 minutes at 37

Summary

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.