This article describes a method for transferring mitochondria from donor cells to modified glioblastoma stem cells (GSCs). The process involves the use of fluorescent labeling to visualize the successful transfer and internalization of mitochondria.
Start with a tube containing deep-red fluorescently labeled donor mitochondria.
Add a cold proliferation medium to disperse the mitochondria.
Dispense the mitochondrial solution near the bottom of the well containing modified glioblastoma stem cells or GSCs.
These modified brain cancer cells are labeled with a blue fluorescent dye and contain red-fluorescently labeled endogenous mitochondria.
The suspension covers the entire surface of the well, uniformly distributing the mitochondria.
Centrifuge the culture plate to bring the mitochondria closer to the cells for their interaction.
Now, incubate the culture plate.
The cell membrane forms large vesicles that engulf extracellular material, including the donor mitochondria.
The cells then internalize these vesicles, releasing the donor mitochondria into their cytoplasm, potentially altering GSC metabolism and functions.
Observe the cells under a confocal microscope.
After image analysis, the presence of blue cells containing green donor mitochondria along with red endogenous mitochondria confirms successful mitochondrial transfer.
To transfer isolated MSC mitochondria to GSCs, add 200 microliters of precooled GSC proliferation medium to the mitochondrial pellet. Dilute the mitochondrial preparation in GSC proliferation medium to consistently allow the addition of 20 microliters of mitochondrial suspension to the GSCs.
Next, slowly add the mitochondria close to the bottom of the well. Centrifuge the plates at 1,500 times G at 4 degrees Celsius for 15 minutes. Then, immediately place the cultures at 37 degrees Celsius. Finally, carry out analysis of mitochondria transfer, FACS analysis, and confocal imaging according to the text protocol.
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