Begin by aspirating the media from the plates containing adherent raw 264.7. Add 10 milliliters of PBS per 15 centimeter plate. Use a cell scraper to detach the cells and pull them into a single 50 milliliter tube.
Pipette the cell suspension up and down to homogenize. Next, transfer 5%of the cell suspension volume into a new 1.5 milliliter tube. Centrifuge at 300G for five minutes at room temperature.
Discard the supernatant and place the total cell fraction pellet on ice. After centrifuging the remaining suspension as described previously, aspirate the supernatant and re-suspend the cell pellet in five milliliters of ice cold mitochondria buffer. Centrifuge the suspension at 300G for five minutes at four degrees Celsius.
Re-suspend the pellet in 0.5 milliliters of cold mitochondrial buffer. And transfer the suspension to a 1.5 milliliter tube. Next, homogenize the suspension, bypassing it 30 times through a 25 gauge needle fitted to a one milliliter syringe.
Then add one milliliter of cold mitochondria buffer to the tube and mix by inversion. After centrifuging the cells, transfer one milliliter of the supernatant to a fresh tube on ice. Re-suspend the pellet in the buffer and homogenize the pellet by passing through a 25 gauge needle fitted to a one milliliter syringe.
Pull the homogenized cell pellets and the supernatant from the previous steps into a single 1.5 milliliter tube. Centrifuge it at 2, 000G for five minutes at four degrees Celsius. Collect the the supernatant and divide it into four 1.5 milliliter tubes.
Make up the volume in each tube to 1.5 milliliters using mitochondrial buffer. Invert the tubes to mix their contents and then centrifuge the tubes. Carefully remove the supernatant and the upper white cellular pellet.
Keep one of the four tubes containing the brown mitochondrial pellet on ice as a crude mitochondrial fraction. Pull the remaining pellets into a new tube and make the final volume one milliliter with mitochondria buffer. Using this protocol, total cells and crude mitochondrial fractions were obtained from the raw 264.7 macrophage cell line and BMDM.
Immuno blood analysis showed that the mitochondrial citrate synthase was enriched in the crude fraction. Proteins from cytosol, plasma membrane, nucleus, lysosome, and endoplasmic reticulum disappeared. Proteome analysis showed an enrichment of more than tenfold in the crude fraction.
The other six cellular compartments were depleted during purification.
