We serve the organization of cycling KP micro domains with a focus on elucidating how these compartments are formed and sustained. We seek to uncover the key components that enable cells to discern spatial signals within these domains, exploring variation in their interpretation and how these features are altered in diseases like asthma and COPD. Infecting primary cells using the Beckman vector is challenging.
For this, the condition should be optimized for individual cell's type in order to achieve sufficient expression of the cAMP biosensor. This protocol allows the real time detection and monitoring ranging from minutes to hours and measurement of cyclo cAMP P dynamics within living cells. This biosensor has high specificity for abiding to cyclo cAMP P.Consequently, the signals generated by this assay directly correlates to cyclo cAMP P levels, significantly reducing the potential interference from the other cellular components and providing reliable results.
To begin, obtain human airway smooth muscle or HASM cell culture. Using a serological pipette, remove and discard HASM media from the culture flask. To wash the cells, add five milliliters of magnesium and calcium free Delbecos PBS, or DPBS, prewarmed to 37 degrees Celsius and gently tilt the flask.
For detaching the cells, add three milliliters of 0.25%tripsin EDTA, and incubate the cells for three to five minutes. After adding five milliliters of fresh media containing antibiotics, transfer the cells to a 15 milliliter tube and centrifuge at 200 G for five minutes. Remove the supernatant and gently add 500 microliters of DPBS without magnesium and calcium along the wall of the tube.
After removing the liquid, count the cells microscopically to adjust the cell density to 40, 000 cells per milliliter. Prepare a master mix containing the vector, reagents, and required number of cells. Dispense 500 microliters of master mix to each well of a 35 millimeter dish and incubate it.
On day two, aspirate the media from each well carefully and add 450 microliters of DPBS containing magnesium and calcium, pre-warmed to 37 degrees Celsius. Wrap the culture plate in aluminum foil and incubate at 37 degrees Celsius for 30 minutes to one hour. Microscopically, verify the cell health and perform serial dilutions to prepare 100 micromolar forskolin and 100 nano molar isoproterenol for the pharmacological treatment.
To begin, culture the vector transformed human airway smooth muscle, or HASM cells. For imaging, turn on the central hub and the inverted fluorescence microscope. Launch the designated software and select the time-lapse capture feature for recording.
Mount the culture dish on the microscope stage. Using auto focus and auto brightness, get a picture of individual cells. Adjust the black balance and excitation wavelength.
Then click autofocus. After finding an area of view with several cytoplasmic fluorescing cells, set the time lapse capturing to 30 seconds for a total period of 20 minutes. Then click okay to begin the data acquisition.
After three minutes, gently add 50 microliters of the drug to the appropriate well, and let the experiment run for 17 more minutes. Open the image files captured during the experiment. Using the polygon or circle tool, select the region of interest of each cell and perform the analysis.
Then open the analyzed data in an Excel sheet and calculate the average brightness for each condition in each photo, starting at the picture just before adding the pharmacological agent. After calculating the change in fluorescence from the initial fluorescence for each average value in each condition, load the values into a statistical analysis software and plot delta F by F zero against time in seconds. As compared to the control, an observable decrease in fluorescence intensity over time was observed upon stimulation of HASM cells with 10 micromolar forskolin and 10 nanomolar isoproterenol.
