Mitochondria Isolation Kit, human From Miltenyi Biotec

Subcellular fractionation, e.g. isolation of mitochondria, is typically performed by density gradient centrifugation of cell lysates. This traditional technique is both time-consuming and labor-intensive. As an alternative approach, differential centrifugation is often used. It is a faster method, however, it results in decreased mitochondria purity compared to density gradient centrifugation. Therefore, a fast, simple and sensitive method is needed. Using magnetic bead technology combined with a specific antibody against the translocase of the outer mitochondrial membrane 22 of human mitochondria, the isolation and elution of human mitochondria is very specific and easy to achieve.

The application of the Mitochondria Isolation Kit, human from Miltenyi is to isolate intact, vital mitochondria from human cells or tissue. It comes with all the necessary reagents for 25 separations, each with up to 10⁷ cells (or appropriate amount of tissue). The kit includes the lysis buffer, a separation buffer concentrate, storage buffer, LS columns for the magnetic separation and Anti-TOM22 MicroBeads, beads conjugated to monoclonal anti-TOM22 antibodies (mouse IgG₁). Some materials for the isolation are needed in addition. For lysis of the cells, either different needles or a Dounce homogenizer might be necessary. For the separation, one of the Miltenyi separation magnets is needed (MidiMACS Separation Unit, #130-042-302, or QuadroMACS Separation Unit, #130-090-976). This is one of the downsides of the kit since it initially adds significant cost to the separation process.

After determining the optimal cell concentration and total cell number to be used for separation and after establishing a reliable lysis protocol, we used the kit for isolation of mitochondria from human platelets. The purity of the preparation was tested using a microscope. The mitochondria were spun onto a coverslip and stained for mitofilin (using an antibody from Mitosciences). To control for cell debris, the slide was counterstained with wheat-germ-agglutinin (WGA, Invitrogen), which stains sialic acid residues and therefore, all double lipid membranes. In addition, using PCR, the mitochondrial fraction was tested for the expression of mitochondrial DNA encoded genes as well as nuclear encoded genes as a control.

After harvesting the cells, they were lysed in the included lysis buffer using techniques empirically tested by the user, resulting in cell disruption but not mitochondrial lysis. The cell lysate was transferred into ice-cold separation buffer. After the addition of the Anti-TOM22 MicroBeads, the lysate was incubated for 1 hour in the refrigerator with gentle shaking. For the magnetic separation, the labeled cell lysate was loaded on a pre-rinsed LS column which was exposed to a strong magnetic field. The magnetically labeled mitochondria were retained within the column. The unlabeled organelles and cell components ran through. The column was washed a couple of times. By removing the column from the magnetic field, the magnetically retained mitochondria can be eluted by simply flushing the column. If the mitochondria are not immediately used for downstream applications, they can be pelleted, resuspended in the storage buffer and kept on ice.

Taken everything together, I think the kit is great for mitochondrial isolation. Since the magnet and the magnet stand are needed in addition to the kit, it is on the more pricy end of isolation kits.