Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy

The overall goal of this procedure is to acquire time-lapse images of primary rat neonatal cardiomyocytes following lentiviral and adenoviral transduction using confocal spinning disc microscopy. This is accomplished by first harvesting cardiomyocytes from rat pups and plating the cells on glass bottom dishes. The next step is to perform transduction of EGFP tagged Conexion 43 by lentivirus, followed by transduction of dominant negative fgfr, one by adenovirus in the cardiomyocytes.

The final step is to acquire time-lapse images using confocal spinning disc microscopy. Ultimately, a movie that is played back from a acquired time-lapse images can be obtained. That shows distribution of EGFP tagged protein expressed by lentiviral genes in live primary cardiomyocytes under altered cell conditions by an adenoviral expressed protein dominant negative fgfr one.

One of the main advantages of this technique is that you can do live cell time lapse imaging using a spinning dis confocal microscope and a desired condition following gene expression induced by adenoviral or then developed transaction. So this method can provide insight into right neonatal cardiomyocytes. It can also be applied to other systems such as primary mouse, neonatal cardiomyocytes, primary right arm mouse, adult cardiomyocytes, and other cultured cells.

Generally, individuals new to microscopy will struggle to take well-focused images due to a slight drift in the stage over time. By using the machine's auto-focus function, stable focus can be maintained and this allows researchers to take well-focused images more easily. The neonatal cardiomyocytes used in this procedure are isolated from one to two day old rat pups.

Remove the beating heart as quickly as possible from the euthanized animal with sterilized tools and place the heart in 30 milliliters of ice. Chilled C-M-F-H-B-S-S in a 50 milliliter conical tube. Typically a total of 10 hearts are collected in two 50 milliliter conical tubes, each with five hearts in 30 milliliters of ice chilled C-M-F-H-B-S-S for filming purposes, two to three hearts are collected.

Swirl the tube to rinse the hearts. All procedures should be done on ice and in a laminar flow hood. From here on, discard the supernatant and transfer the hearts to a new sterilized 10 centimeter plastic dish on ice.

Be careful not to aspirate the hearts with the aspirator. Remove large vessels and or undesired tissues from the hearts. Add 10 milliliters of ice chilled C-M-F-H-B-S-S to the dish to rinse the hearts.

Next, transfer all hearts to a new sterilized 10 centimeter plastic dish on ice. Mince the hearts with scissors to less than one cubic millimeter. Add nine milliliters of chilled C-M-F-H-B-S-S to the minced hearts.

Add one milliliter of chilled trypsin. Reconstituted with C-M-F-H-B-S-S. Seal the dish with paraform.

Cover with aluminum foil and place it in a cold room overnight on the following day, transfer all heart tissue with buffer to a 50 milliliter conical tube on ice. Add one milliliter of trypsin inhibitor in C-M-F-H-B-S-S. Cap the 50 milliliter conical tube tightly to prevent contamination.

Incubate the tube for about 30 minutes in a 37 degrees Celsius incubator to warm the tissue and buffer to 30 to 37 degrees Celsius. Next, add five milliliters of collagenase in liebovitz L 15 and incubate at 37 degrees Celsius for 45 minutes with gentle shaking after 45 minutes. Tritrate tissues using an auto pipette pipette 20 times at a speed of three milliliters per second.

Allow tissue residues to settle for three minutes and then filter the supernatant with a 70 micron cell strainer. Add five milliliters of the Leibovitz L 15 to the remaining tissue clumps and tri rate and filter the snat again. Wash the cell strainer with two milliliters of the Leibovitz L 15.

Place the filtered cell solution in a hood for 20 to 60 minutes to allow the collagenase to digest the partially degraded collagen sediment. The cells at 100 times G for five minutes. Resuspend the cells in 20 milliliters of DMEM containing 10%fetal bovine serum and 20 units per milliliter.

Penicillin streptomycin prelate the cells on two 10 centimeter plastic cell culture dishes for one hour at 37 degrees Celsius in a CO2 incubator for cardiomyocyte selection, fibroblasts will attach more readily to the bottom of the dish than cardiomyocytes After one hour, swirl the dishes gently and collect the cardiomyocyte containing supernatant from the dishes. Sediment the cells at 100 times G for five minutes. Resuspend the cells in DMEM containing 10%FBS 10 units per milliliter, penicillin streptomycin and 0.1 millimolar bromo deoxy uridine plate cells at two times 10 to the fifth cells per dish in gelatin coated 3.5 centimeter glass Bottom dishes for observation using the microscope incubate in the CO2 incubator at 37 degrees Celsius.

Do not disturb the cells after plating for at least 24 hours prior to lentiviral transduction of the cardiomyocytes. Lentiviral plasmids are packaged and lentiviral solution is collected according to the procedures described in the accompanying manuscript. To begin the procedure for lentiviral transduction, remove medium from a 3.5 centimeter glass bottom dish of rat neonatal cardiomyocytes and add one milliliter of filtered lentiviral solution.

Incubate the dish in a CO2 incubator at 37 degrees Celsius for six hours. After six hours. Replace the medium with new DMEM, with MEM containing 5%FBS and penicillin streptomycin incubate in the CO2 incubator at 37 degrees Celsius for three days.

Integration of the exogenous gene into the host genome by lentivirus is considered to be complete by 72 hours prior to adenoviral transduction. The adenoviral stock solution must be titrated as described in the accompanying manuscript. Retrieve the 3.5 centimeter glass bottom dish of rat neonatal cardiomyocytes that underwent lentiviral transduction.

Replace the medium with 750 microliters of diluted adenoviral solution. Reduction of medium volume at transduction to about 50%of the original volume is critical for high transduction efficiency. Incubate in the CO2 incubator at 37 degrees Celsius for four to eight hours.

Next, replace the medium with fresh medium incubate for 24 to 48 hours before using the cells for live cell imaging experiments. Image acquisition, using a confocal spinning disc microscope with a temperature controlled chamber and a CO2 environmental system will be demonstrated at least one hour prior to imaging. Turn the temperature control system on so that all devices equilibrate to 37 degrees Celsius.

Turn on the confocal spinning disc microscope system. Place the 3.5 centimeter glass bottom dish containing rat neonatal cardiomyocytes on the stage of the confocal spinning disc microscope in a temperature controlled chamber. Turn the shutter for the fluorescent light source on find cells expressing fluorescent proteins under the microscope through the eyepiece.

Turn off the shutter for the fluorescent light source. Change the light path to the spinning disc confocal microscope. Adjust settings for acquisition appropriately using the operating software checking images displayed on the screen set settings for the fluorescent image acquisition.

For example, select change channels using light paths and channel one EGFP to acquire fluorescent signal from EGFP for channel one. Set the frequency between time points by configuring time-lapse settings in order to take time-lapse images using the operating software. For example, to acquire one time point every five minutes, select minutes per time point from the dropdown menu and enter five into the text field.

Start the time lapse acquisition by clicking the start button to acquire an image sequence. At the completion of imaging acquired, images can be played back as a movie file and analyzed. Using the analysis software, select the acquired images to be included in a movie if necessary.

Crop regions of interest from images and select interesting time points to reduce the file size of the movie, export images as a movie file in a VI or MOV format. Select a movie format from format and the timing required for the finished movie. Then click export.

In this representative experiment, rat neonatal cardiomyocytes were transduced with a lentivirus encoding EGFP tagged CONEXION 43 or a mutated CONEXION 43, and an adenovirus encoding a dominant negative fibroblast growth factor receptor one FGFR one dn. The expression of EGFP tagged proteins was confirmed by confocal spinning disc microscopy while the of the dominant negative fibroblast growth factor receptor was confirmed by immunochemistry and western blot time-lapse images of cardiomyocytes were acquired and played back as a movie. In this example, the images moved in the XY plane possibly because the temperature equilibration was not adequate or the multiple acquisition mode was not functioning properly.

This is from time-lapse images of cardiomyocytes expressing the mutated Conexion 43 and FGFR one dn. Here, the temperature equilibration was adequate and only a single region of interest was acquired, which resulted in images without movement in the XY plane. The beating of cardiomyocytes was assessed after imaging in order to confirm cell viability.

This movie shows that after 16 hours of time-lapse imaging, EGFP expressing cardiomyocytes maintained their functional properties and beat rhythmically. With this technique, you can do live imaging of primary ator cardiomyocytes. Please remember that working with adenovirus and lentivirus can be hazardous and precautions such as the contamination should always be taken when working with these viruses.