Once around 25 million mitochondria donor cells are obtained on day seven, harvest the exponentially grown cells in a 50-milliliter tube and collect them by centrifuging at room temperature and 520g for five minutes. Wash the cells with cold phosphate-buffered saline and sediment them by centrifugation at room temperature in 520g for five minutes. Now onwards, perform the whole mitochondrial extraction at four degrees Celsius using cold reagents and keep the cells or mitochondria on ice.
After the third centrifugation, discard the supernatant by aspiration using a glass pipette coupled to a vacuum pump and resuspend the packed cells in a volume of hypotonic buffer equal to seven times the cell pellet volume. Then transfer the cell suspension into a homogenizer tube and let the cells swell by incubating them on ice for two minutes. Break the cell membranes by performing 8 to 10 strokes in the homogenizer coupled to a motor-driven pestle rotating at 600 rotations per minute.
Into the cell homogenate, add the hypotonic buffer equal to seven times the volume of the initial pellet to generate an isotonic environment. Transfer the homogenate into a 15-milliliter tube and centrifuge it in a fixed rotor at 1, 000g and four degrees Celsius for five minutes. Then collect only 3/4 of the supernatant, leaving a large margin from the pellet to avoid contamination with nuclei or intact cells and transfer it to another tube.
After repeating the process twice, transfer the supernatant containing the mitochondrial fraction into 1.5-milliliter tubes. Centrifuge the tubes at the maximum speed of 18, 000g for two minutes at four degrees Celsius. Discard the supernatant and wash the mitochondria-enriched pellet with buffer A.Combine the content of the two tubes into one and centrifuge as demonstrated before.
Repeat the process until all the material is in only one tube. Wash the pellet obtained from the last centrifugation using 300 microliters of buffer A.Quantify the mitochondrial protein concentration using the Bradford assay. Before fusion with the mitochondrial recipient cell line, evaluate the absence of nuclei contaminants in the mitochondrial fraction by immunodetection of nuclear proteins.
Alternatively perform quantitative polymerase chain reaction or QPCR amplification of a nuclear gene.
