Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

The overall goal of this procedure is to simultaneously map transmembrane potentials and calcium transients in HL one atrial monolayers loaded with both calcium and voltage dyes. This is accomplished by first culturing the HL one cells on a cover slip to a proper confluent density. The second step is to load the calcium and voltage sensitive dyes into the cells.

Next, the HL one monolayer is stimulated with a bipolar electrode. The final step is to record the action potential and the calcium transient signals by fluorescence microscopy. Ultimately, the action potential and calcium transient traces can be compiled to show propagation of the signals across the confluent monolayer over time.

This method can help answer key questions in the cardiac electrophysiology field, such as how calcium, transients, and action potentials are related. In the electrophysiological properties of cultured HL one cells subjected to various drug treatments, Though this method can provide insight into patterns of action, potential and calcium transient propagation and microscopic level. It can also be applied to biological systems such as pathologically remodeled human hearts and arrhythmogenic animal models.

Generally, individuals new to this method while struggle because hhl one cells maintaining cardiac mouse phenotypes are difficult to culture and are carefully optimize. The protocol is needed, the culture passenger and the steam monolayer. To begin make all of the stock solutions for the experiment as indicated in the text protocol, grow the HL one cells in T 25 flasks and replace the medium with five milliliters of fresh supplemented claycomb medium daily.

The cells should be ready to passage at day 5 24 hours before the HL one cells reach co fluency. Place round 20 millimeter cover slips into the wells of a 12 well culture plate. Add one milliliter of the gelatin fibronectin solution to each well to coat the cover slips, and then incubate the plate overnight at 37 degrees Celsius.

The next day aspirate the gelatin fibronectin solution from the wells of the culture plate and then add 1.5 milliliters of supplemented final clay home medium to each well. Next, aspirate the medium from the T 25 flask containing the HL one cells, and then rinse the culture briefly with PBS at 37 degrees Celsius. Aspirate the PBS from the flask and add three milliliters of the trypsin EDTA solution to the bottom of the T 25 flask so as not to disturb the cells gently lower and rotate the flask to cover the cells with the solution, and then place the flask in the incubator for one minute at 37 degrees Celsius.

Next, carefully aspirate the tripsin EDTA solution from the culture so as not to disturb the cells. And then add an additional 1.3 milliliters of the tripsin EDTA solution to the flask. Incubate the flask at room temperature for one additional minute.

Observe the cells under a microscope to determine whether they have detached from the surface of the flask. Gently tap the flask to dislodge any cells that remain attached. Once all of the cells have detached, add 1.3 milliliters of the soybean tripsin inhibitor solution to the cells, and then transfer the cells to a 15 milliliter centrifuge tube.

Rinse the empty flask with five milliliters of the wash medium and add the rinse to the cells in the tube. Centrifuge the tube at 500 times G for five minutes, and then aspirate the S supernatant without disturbing the cell pellet. Gently resuspend the cell pellet in six milliliters of supplemented medium.

Add 0.5 milliliters of the cell suspension to each well of the culture plate. Incubate the plates at 37 degrees Celsius, changing the medium daily until the cultures are confluent. To prepare the working dye solutions dilute the road two and the RH 2 37 stock solutions by one to 1000 in Hank's salt solution.

Observe the 12 well culture plates under a microscope to determine whether the HL one cells are confluent. Usually four days after seeding when the cells are confluent, gently remove the cover slip from the well and rinse the cover slip with Hank's salt solution. Place the cover slip into a 35 millimeter Petri dish with three milliliters of road two working solution.

Put the Petri dish into a 37 degrees Celsius water bath and bubble, a continuous stream of gas through the solution for oxygenation. Incubate for 30 minutes to stain the cells with the calcium sensitive dye. Then transfer the cover slip to three milliliters of oxygenated RH 2 37 solution and incubate for 15 minutes.

The cell monolayer should now be stained with both calcium and voltage sensitive dyes. To prepare the cells for imaging, place the cover slip with the double stained cell monolayer in a circulation chamber on an inverted fluorescence microscope. Adjust the flow speed through the chamber to maintain the temperature at 35 degrees Celsius with a gradient of no more than 0.3 degrees Celsius across the chamber.

Flo Hank salt solution across the cell monolayer for five minutes to remove any excess dye. After washing the monolayer, place a bipolar electrode in the solution as close as possible to the monolayer without touching the cells. Test the health of the monolayer according to the text protocol.

To paste the monolayer. Program the electrode to administer pulses of a two millisecond width for up to 500 milliseconds per cycle. While pacing the monolayer image, the cells with a 40 x objective and excite the calcium and voltage sensitive to eyes.

Using an LED light source coupled to an excitation filter and a dichroic mirror, pass the emitted fluorescence through a dichroic mirror to split the calcium and voltage sensitive signals and collect the fluorescent signals separately with two CCD cameras. Image four different areas of the monolayer to obtain complete coverage and be careful to record the signal for less than two seconds on each field. To avoid photobleaching.

A confluent HL one monolayer exhibits a regular contraction rhythm when pasted by the electrode. The voltage and calcium signals were recorded for a single electrically paced beat of the HL one monolayer in a 500 millisecond cycle onset of the voltage signal occurs shortly before the calcium transient. While the calcium signal persists longer than the voltage signal surface.

Intensity maps were generated from the voltage and calcium fluorescent signals across the HL one cell monolayer. During a 200 millisecond time course, blue indicates low signal while red indicates high signal. Both signals begin at the left of the monolayer and they propagate across and to the right of the monolayer.

Over time, the action potentials propagate and return to the baseline much more rapidly than the calcium signal Once mass. This to mapping procedure can be done in one hour if it's performed properly. While attempting this procedure, it is important to remember to use the cells that are at optimal density and to use caution when pacing electrodes to avoid damaging the monolayer.

After watching this video, you should have a good understanding of how to prepare an optically map, HO one atrial cell monolayers, using both calcium and voltage sensitive dyes.