The overall goal of this experiment is to display our protocols for the culture and transfection of HEK293 and C2C12 cells with CatchER plus and its application for monitoring ERSR receptor mediated calcium transients in the measurement of the NC2KD. This method can help answer key questions in calcium signaling and the imaging such as the basal calcium concentration and its rapid changes in the ERSR of definite cells and how to apply biosensor CatchER plus for these measurements. The main advantage of this technique is that the fluorescence response of single cells can be recorded in real time with rapid kinetics.
The implications of this technique extend toward diagnosis of ERSR calcium related diseases because CatchER plus can monitor abnormal calcium transients in these organelles that arise from dysfunctional calcium signaling elements. The visual demonstration for this method is critical as the cell transfection and fluorescence imaging steps are difficult to learn because cell culture and microscopy have multiple areas for error. Demonstrating the procedure will be Florence Reddish, a post-doc from our laboratory.
To begin this experiment, seed HEK293 cells onto the sterilized glass microscope slides in the six centimeter dishes and culture for one to two days until they are about 70%confluent on the day of transfection. Next, empty DMEM from the dish and add three milliliters of the reduced serum medium. Then, add DNA transfection reagent mixture to the dish and incubate for four to six hours at 37 degrees Celsius.
Transfection of HEK293 cells is generally easier than other cell lines, but the cells are less resilient to harsh conditions. Therefore, it is critical to only expose them to transfection reagents for four to six hours to avoid apoptosis. After four to six hours, wash the cells with five to six milliliters of HBSS.
Discard the HBSS from the dish and replace with three milliliters of fresh DMEM. Subsequently, incubate the cells for 48 hours at 37 degrees Celsius to allow the expression of GECI. For C2C12 myoblast cells, trypsinize the cells by adding one to two milliliters of trypsin to cover the bottom of the dish.
Next, place the dish in a 37 degrees Celsius incubator for two to six minutes to allow the trypsin to loosen the cells from the bottom of the dish. Once the incubation is complete, remove the trypsin and aspirate the cells in eight milliliters of DMEM. Pipette the cells thoroughly with DMEM to evenly disperse and re-suspend the cells.
Then, seed them onto the sterile coverslips in six centimeter culture dishes and then add three milliliters of DMEM to achieve a final confluence of 60%Following that, transfect the cells on the same day. Incubate them for 24 hours at 37 degrees Celsius. After incubation, wash the cells with five to six milliliters of HBSS.
Discard the HBSS from the dish and replace with three milliliters of fresh DMEM. Then, incubate them at 37 degrees Celsius for 48 hours to allow expression of the GECI. In this procedure, turn on the microscope and light source, then open the simple PCI program.
Using a cotton swab, cover the bottom of the low-profile open-diamond bath imaging chamber with a thin layer of sealant. Then, take a slide containing the transfected cells out of the incubator and gently rinse it three times with around one milliliter of Ringer's buffer with 10 millimolar glucose and 1.8 millimolar calcium preheated to 37 degrees Celsius. Leave roughly one milliliter of Ringer's buffer in the dish to prevent the cells from drying and use forceps to take the slide out of the dish.
Absorb excess solution by touching the edge of the slide to a laboratory tissue. Then, place it onto the side of the chamber with the cells facing towards the grease. Subsequently, secure the chamber onto the stage mount using a screwdriver.
Add around one milliliter of Ringer's buffer to the chamber to prevent the cells from drying while mounting it onto the stage and completing the microscope setup. Switch the microscope objective to the 40 times oil immersion objective. Add one drop of the immersion oil to the objective.
Afterward, place the mount onto the microscope stage. Then, place the vacuum tip in the chamber to maintain the solution leveled and prevent it from overfilling. Next, raise the objective using the course adjustment until the oil on the objective touches the bottom of the slide.
Using the bright field mode, adjust the gain to 175 and the exposure time to 0.03 seconds to focus the cells. Once the cells are focused, switch to fluorescence mode using 488 nanometer excitation and change the exposure time to 0.07 seconds. Locate a field of view that has enough cells that are healthy with adequate fluorescence for imaging.
Then, take pictures in fluorescence and bright field mode. Circle a region of interest in the cells or circle the whole cell to record the intensity from the chosen area. Now, start the intensity measurement with the frame rate set at one every five seconds.
Allow the baseline intensity to stabilize for 20 to 30 frames. Next, calculate the amount of 4-CMC needed from a 20 millimolar stock based on the final chamber volume of 450 microliters to make a final concentration of 200 micromolar. Then, carefully transfer 60 microliters of Ringer's buffer from the chamber to a microcentrifuge tube.
Dilute the calculated amount of 4-CMC with this solution and add back to the chamber to evoke the intended response from the cells being imaged. Allow time for the drug to bind to the receptor and subsequent signal decrease. Then, wash the cells with three to six milliliters of Ringer's buffer while simultaneously adding the event marker.
Repeat the procedures for 100 micromolar ATP and 15 micromolar CPA using a new slide of the transfected cells for each assay. Once the measurement is complete, end the data collection in the intensity measurement window in the simple PCI program. Subsequently, take the fluorescence and bright field pictures of the cells.
Then, open the data file for the experiment. Re-circle the cells or regions of interest to recalculate the intensity values if necessary. To determine the NC2KD and C2C12 myoblast cells, repeat the transfection procedure and add one milliliter of calcium-free Ringer's buffer in the chamber.
Permeabilize the cells with 0.002 to 0.005%saponin and intracellular buffer for 15 to 30 seconds to empty the cells of any CatchER plus that may be in the cytosol. Then, wash the cells with three to six milliliters of KCL buffer containing EGTA and 10 micromolar ionomycin. Allow the intensity to decrease until a plateau is reached.
Following that, rinse the cells with three to six milliliters of KCL buffer with 10 micromolar ionomycin and allow the intensity to stabilize. Afterward, add different calcium solutions to allow the intensity to reach a plateau between each addition. To calibrate the sensor for basal calcium concentration quantification, use EGTA and 100 millimolar calcium buffer to get the minimum and maximum fluorescence intensity.
Shown here is the representative traits for the normalized intensity collected from the permeabilized C2C12 myoblast cells transfected with CatchER plus. Cells were permeabilized with 0.005%saponin in the intracellular buffer for 15 to 30 seconds. EGTA and calcium solutions were prepared in KCL buffer and N refers to the number of cells imaged.
These inset fluorescence images are the representatives of cells before and after treatment. 0.2, 0.6, two, five, 10, 20, 50, and 100 millimolar calcium was added to the permeabilized C2C12 myoblast cells in the presence of 10 micromolar ionomycin to get a KD of 3.1 plus or minus 1.4 millimolar. This figure shows a representative intensity measurement of CatchER plus transfected cells at minimum and maximum values after the addition of one millimolar EGTA and 100 millimolar calcium with 10 micromolar ionomycin each respectively.
Once mastered, this technique can be done in 30 to 60 minutes for the imaging or three days if including the transfection. While attempting this procedure, it's important to remember to always handle the cells in a sterile environment, get reagents ready before the experiment, to apply an even layer of grease to the chamber, and to have your vacuum attached before beginning. Following this procedure, other methods like western blot can be performed in order to answer additional questions like the expression level of CatchER plus.
After its development, this technique paved the way for researchers in the field of calcium signaling and imaging to explore ERSR calcium dynamics in mammalian cells. After watching this video you should have a good understanding of how to transfect cells and image them using CatchER plus.
