Cardiomyocytes में फास्ट कैल्शियम अपशिष्टों को मापने
Summary
हम रहने वाले confocal माइक्रोस्कोपी स्कैनिंग लेसर का उपयोग कर कोशिकाओं में धीमी अपशिष्टों से तेजी microsecond () कैल्शियम की घटनाओं को अलग करने की विधि प्रस्तुत करते हैं. विधि एक सेल में कई सौ पिक्सल के रिकॉर्डिंग लाइन स्कैन द्वारा कैल्शियम संकेतकों के प्रतिदीप्ति तीव्रता उतार चढ़ाव के उपाय. हिस्टोग्राम विश्लेषण हमें अलग कैल्शियम अपशिष्टों के समय तराजू अलग करने के लिए अनुमति देता है.
Abstract
Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase Cβ- Gαq pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated Gαq 7. This entrapment has the effect of stabilizing the activated state of Gαq and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.
Protocol
Discussion
हम एक को देखने और रहने वाले cardiomyocytes में तेजी से कैल्शियम संकेतों को अलग विधि विकसित की है. जबकि इन मापों के प्रयोगात्मक शर्तों अन्य सेल 10 प्रणाली के रूप में के रूप में अच्छी तरह से 11 cardiomyocytes पहले लागू ?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
यह काम राष्ट्रीय स्वास्थ्य अनुदान GM53132 2R01 और GM071558 P50 के संस्थान के द्वारा समर्थित किया गया.
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| DMEM | Invitrogen | ABCD1234 |
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