Electro fusion involves application of short high voltage electric pulses to cells in close context. In this video, we demonstrate electro fusion of cells in vitro by means of modified Adherence.Method. Experiments were performed on mouse Melanoma cells, BF one.
For the experiment, follow these steps. Grow cells in two separate culture flasks to 80%confluence. Separately, add 2.1 microliters of each stock solution.
10 millimolar C-M-F-D-A or CMRA respectively to three milliliters of Krebs Hess Buffer in a centrifuge tube. Rinse cells twice with Krebs Hess buffer and then insert loading solutions into the flasks. Loading solutions contain approximately seven Micromolar C-M-F-D-A or CMRA respectively.
Incubate cells for 30 minutes in a controlled atmosphere. During this first incubation, regions pass freely through cell membranes into the cytosol where they are transformed into membrane IMP permeant reaction products. After this first incubation, rinse and then incubate the cells with culture medium for another two hours.
The cells are observed using a fluorescence microscope equipped with a cooled CCD camera in the acquisition software. Set the excitation wavelength to 548 nanometers and choose an appropriate band pass filter threat fluorescence image of the cells loaded with CMRA for the cells loaded with C-M-F-D-A. Set the excitation wavelength to 492 nanometers and choose a band pass filter for green fluorescence image trypsin eyes, and count the cells in both flasks and mix red and green cells together In a ratio one to one, adjust cell concentration to 5 million cells per milliliter.
Place a 20 microliters drop of cell suspension in the middle of each well. In a 24 multi-well plate incubate cells in a controlled atmosphere for 20 minutes to allow cells to slightly attach to the surface of the well in a monolayer and establish cell contacts between themselves. Place the multi-well with cells onto the microscope stage.
Position the electrodes at the bottom of the well and connect them to the pulse generator To achieve optimal electro fusion and maintain cell viability, adequate parameters of electric pulses should be used. These parameters depend on cell line and should be determined in preliminary experiments. Remove the culture medium and wash the cells with one milliliter ISO omu potassium phosphate buffer at 350 microliters hypos Miller potassium phosphate buffer in order to induce cell swelling.
Leave cells in hyperosmal buffer for two minutes before applying electric pulses. Electric pulses should be applied when cells are close to their maximum volumes. That is before they start regulatory volume.
Decrease in our experiment. A train of eight rectangular pulses with duration of 100 microseconds at one hertz was applied. After pulse Delivery.
Leave the cells undisturbed for 10 minutes after 10 minutes. Determined Diffusion yield by means of face contrast and fluorescent microscope. E acquire three images, face contrast, red and green fluorescence at five randomly chosen fields of view.
In each, well create three channel images from each image triplet in image J software in order to improve visual quality of images to pre-processing steps can be applied to the original images, background, subtraction, and contrast enhancements with default parameter sets. Finally, three channel images are composed using RGB to gray image J plugin. In such image, the resting cytoplasm can be seen together with cell membranes.
Thus few cells can easily be determined in the three channel image count all three types of cells, red, green, and dually fluorescent. Determine the percentage of dually fluorescent cells by dividing the number of dually fluorescent cells with a number of all cells in each image. Fusion yield is then defined as the percentage of dually fluorescent cells multiplied by two.
Since half of the view cells are not detected when cells of the same color views.
