Rapid and easy semi-quantitative evaluation method for diacylglycerol and inositol-1,4,5-trisphosphate generation in orexin receptor signalling.
OBJECTIVE
Fluorescent protein-based indicators have enabled measurement of intracellular signals previously nearly inaccessible for studies. However, indicators showing intracellular translocation upon response suffer from serious limitations, especially the very time-consuming data collection. We therefore set out in this study to evaluate whether fixing and counting cells showing translocation could mend this issue.


METHODS
Altogether three different genetically encoded indicators for diacylglycerol and inositol-1,4,5-trisphosphate were transiently expressed in Chinese hamster ovary cells stably expressing human OX(1) orexin receptors. Upon stimulation with orexin-A, the cells were fixed with six different protocols.


RESULTS
Different protocols showed clear differences in their ability to preserve the indicator's localization (i.e. translocation after stimulus) and its fluorescence, and the best results for each indicator were obtained with a different protocol. The concentration-response data obtained with cell counting are mostly comparable to the real-time translocation and biochemical data.


CONCLUSIONS
The counting method, as used here, works at single time point and looses the single-cell-quantitative aspect. However, it also has some useful properties. First, it easily allows processing of a 100- to 1000-fold higher cell numbers than real-time imaging producing statistically consistent population-quantitative data much faster. Secondly, it does not require expensive real-time imaging equipment. Fluorescence in fixed cells can also be quantitated, though this analysis would be more time-consuming than cell counting. Thirdly, in addition to the quantitative data collection, the method could be applied for identifying responsive cells. This might be very useful in identification of e.g. orexin-responding neurones in a large population of non-responsive cells in primary cultures.
Ekholm
M E
ME
Unit of Physiology, Department of Neuroscience, Uppsala University, Uppsala, Sweden.
Johansson
L
L
Kukkonen
J P
JP
D000818:Animals; D001692:Biological Transport; D002452:Cell Count; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D003600:Cytosol / Q000378:metabolism; D003952:Diagnostic Imaging; D004075:Diglycerides / Q000096:biosynthesis*; D005240:Feasibility Studies; D005260:Female; D005404:Fixatives; D005453:Fluorescence; D006801:Humans; D007202:Indicators and Reagents / Q000493:pharmacokinetics; D015544:Inositol 1,4,5-Trisphosphate / Q000096:biosynthesis*; D008722:Methods; D064446:Orexin Receptors; D009994:Osmolar Concentration; D010053:Ovary / Q000166:cytology / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D018013:Receptors, Neuropeptide / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D013997:Time Factors; D014018:Tissue Distribution
D004075:Diglycerides; D005404:Fixatives; D007202:Indicators and Reagents; D064446:Orexin Receptors; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; D015544:Inositol 1,4,5-Trisphosphate