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Measurement of heat production (thermogenesis) in cells using ERthermAC dye

CW Chih-Hao Wang
YT Yu-Hua Tseng

Last updated date: Sep 29, 2020 Views: 1550 Forks: 0

original An abbreviated version of this protocol was published in Science Translational Medicine in Aug, 2020
CRISPR-engineered human brown-like adipocytes prevent diet-induced obesity and ameliorate metabolic syndrome in mice
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Measurement of heat production (thermogenesis) in cells using ERthermAC dye

 

Materials

·        BioTracker ERthermAC Temperature-Sensitive Live Cell Dye (SCT057, Merck)

(https://www.merckmillipore.com/TW/zh/product/BioTracker-ERthermAC-Temperature-Sensitive-Live-Cell-Dye,MM_NF-SCT057?ReferrerURL=https%3A%2F%2Fl.facebook.com%2F&fbclid=IwAR1xNHOYdLL4iTg8IRPS0fHiwa88ZmXFFCu2vYnZTVg4ivkjz3_YKd1AFb8)

 

·        Prepare stock solution of ERthermAC dye (1 mM in DMSO)

 

Procedures

1.     Differentiate brown/white adipocytes in a black 96-well plate with clear bottom  

2.     Prepare 250 nM ERthermAC dye in DMEM-H serum-free medium

3.     Add 100 uL of staining solution per well

4.     Incubate 37oC for 30 min

5.     Wash with PBS twice

6.     Add 90 uL of DMEM-H phenol red-free medium each well

7.     Equilibrate plate at 25 oC

8.     Measure 2-3 points of basal fluorescence (ex/em, 543/590) in a microplate reader

9.     Add PBS or 10 uL of 10 mM CL-316,243 (CL)/forskolin (Fsk) each well (final 1 mM)

10.  Measure the fluorescence (ex/em, 543/590) every 5 min up to 1.5 ~ 2 hr.

11.  Calculate the decrease of fluorescence over time after drug treatment

 

 

 


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How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Wang, C and Tseng, Y(2020). Measurement of heat production (thermogenesis) in cells using ERthermAC dye. Bio-protocol Preprint. bio-protocol.org/prep525.
  2. Wang, C., Lundh, M., Fu, A., Kriszt, R., Huang, T. L., Lynes, M. D., Leiria, L. O., Shamsi, F., Darcy, J., Greenwood, B. P., Narain, N. R., Tolstikov, V., Smith, K. L., Emanuelli, B., Chang, Y., Hagen, S., Danial, N. N., Kiebish, M. A. and Tseng, Y.(2020). CRISPR-engineered human brown-like adipocytes prevent diet-induced obesity and ameliorate metabolic syndrome in mice . Science Translational Medicine 12(558). DOI: 10.1126/scitranslmed.aaz8664

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