We studied our channels related to temperature sensation, age sensation, and the pain sensation. We are interested in screen small molecules activities in our channels and the study of their mechanisms. We have screened two TRPV1 activating agonists and conducted a detailed study on their mechanisms of action.
This technology enables real-time, quantitative, and simultaneous monitoring of calcium changes in multiple cells, making it a simple and rapid approach for research applications. We aim to explore traditional Chinese medicines treatment mechanism for the various disease through the modernation of calcium-related ion channels. To begin, prepare sterile glass slides with eight millimeter diameter and place them in a 24 well plate.
Add 500 microliters of Poly-D-lycine buffer to each well. Incubate the slides at 37 degrees celsius for one hour to allow coating. Then using a pipette, discard the coating solution from each well.
Wash the slides once with Dulbecco's PBS and set the washed slides aside for later use. Seed the cells onto the prepared 24 well plate at a density of approximately 1.5 times 10 to the power of 5 cells per well, and place the plate in the incubator until the cells become adherent. Next, mix the Fura-2/AM stock solution and Pluronic F-127 with Hank's buffer containing 1.3 millimolar calcium ions.
Using aluminum foil, cover the Fura-2/AM working solution to protect it from light. For pretreating the cells, transfer the glass slides with the cells to a new 24 well plate containing Hank's buffer. Using a pipette, discard the buffer from each well and add 500 microliters of Fura-2/AM working buffer to each well.
Incubate the plate at room temperature in the dark for 30 minutes to allow probe loading. Then remove the Fura-2/AM working buffer from each well and wash the cells three times with Hank's buffer to remove excess Fura-2/AM. To begin, obtain the pretreated cells for single cell calcium imaging and start all the components of the fluorescence microscope.
Open the fluorescence imaging software from the available options. Select the required protocol and click on Okay. Then, choose New Experiment from the main menu to initiate a fresh experimental setup.
Now, mount the perfusion chamber on the microscope stage and carefully place the Fura-2/AM treated cell slides in the chamber containing Hank's buffer solution. Adjust the focus under white light for optimal viewing. Select Focus on the experiment control panel.
Choose the desired wavelength for focusing such as 380 nanometers and click on Start Focusing to initiate focal adjustments. Adjust the focus on the cells using Rough Focus to bring the objective close, followed by Find Focus for precise adjustments. Observe the expression of the target protein by wavelengths of FITC or TRITC.
Then click Stop Focusing and close the focus panel. Click on Configure Experiment in the task bar and select the desired floors for imaging in the configuration settings. Now click on the Region button in the menu bar to define areas for imaging and choose the Desired Illumination type for imaging.
Click on Acquire Images, then press Okay to capture images. Identify the desired cells expressing the target protein under fluorescence, considering the blank reading with the controls under 380 nanometers. To undo a selected region, right click on the circle and select Delete Region.
Open the References menu, check the box for Subtract References and then click Okay to apply background subtraction. On the Experiment control panel, click on Log Data to record the experiment data. In the Time Lapse section of the control panel, set the Data Acquisition Interval to one second.
Then click Zero Clock and Acquire on the Experiment control panel to begin data acquisition. Now apply treatments to the cells as per the requirements. Once data acquisition is complete, click on Pause to halt the process.
Save the acquired data and proceed with the analysis. To end the experiment, click on File and select Close Experiment. Finally, close the software and transfer all saved data from the computer.
Primary keratinocytes loaded with the Fura-2 probe displayed visible cell morphology at 380 nanometers wavelength. The thermal response of keratinocytes showed an increase in intracellular calcium ion concentration during heating with a similar response observed during cooling. The thermal response was abolished in keratinocytes, lacking the STIM1 gene indicating the gene's role in thermal calcium signaling.
HEK293T cells over expressing STIM1/Orai1 showed a notable thermal response upon cooling confirming successful gene expression as evidenced by green and red fluorescence markers. The store operated calcium entry response in the cells over expressing STIM1-Orai1 was effectively induced by sequential changes in extracellular calcium concentration and Cyclopiazonic Acid edition via profusion. Direct pipetting of capsaicine, a TRPV1 agonist, triggered extracellular calcium influx in the cells expressing the TRPV1 plasmid.
