Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

The overall goal of this procedure is to rapidly isolate pure and respiration competent mitochondria from rat tissue. This is accomplished by first associating and homogenizing tissue samples. The next steps are to mix subin A into the homogenate and incubated on ice.

This is followed by filtering the homogenate mixing in BSA and filtering it again. The final steps are to spin down the filtrate and resuspend the mitochondrial pellets in respiration buffer. Ultimately, results can show that isolated mitochondria are viable and respiration competent through respiration analysis and an A TP luminescence assay.

The main advantage of this technique over existing methods that report mitochondrial isolation times ranging from 60 to a hundred minutes, is that viable and respiration competent mitochondria can be isolated in less than 30 minutes. Allison mackay, A technician from a collaborating lab will be demonstrating the A TP luminescence assay Just before beginning. Dissolve the subin A in one milliliter of homogenizing buffer and dissolve the BSA also in one milliliter of homogenizing buffer.

Now start with collecting two fresh tissue samples using a six millimeter biopsy sample punch, and store them in one XPBS on ice in a 50 liter centrifuge tube. When ready, transfer the tissue to a dissociation C tube containing five milliliters of ice cold homogenizing buffer. Then homogenize the tissue by fitting the tube to the tissue dissociation and selecting the preset mitochondrial isolation cycle.

When completed, put the tube back on ice and add 250 microliters of sub to lyin a stock solution. Mix this up by inversion. When the mixture looks uniformly distributed, incubate the homogenate on ice for 10 minutes.

Next, put a 40 micron mesh filter on a 50 milliliter conical centrifuge tube and pack this into the ice. Moisten the filter with homogenizing buffer and discard the buffer into a waste container. Then once all is ready, pour the homogenate through to the filtrate.

Add 250 microliters of BSA stock solution and mix it in by inversion. However, this step should be skipped for mitochondrial protein analysis. Now prepare two more.

50 milliliter conical centrifuge tubes on ice fit one with a 40 micron filter and the other with a 10 micron filter. Wet both filters with a little homogenizing buffer and discard this buffer. Then pour the homogenate first through the 40 micron filter, and then through the 10 micron filter.

These filtration steps significantly improve the purity of the mitochondria. Now divide the filtrate between two pre chilled 1.5 milliliter micro fuge tubes, and centrifuge them at 9, 000 Gs for 10 minutes at four degrees Celsius. Then remove the supernatant and resuspend and combine the two purified mitochondrial pellets.

In one milliliter of ice cold respiration buffer, it is vital that the entire procedure be conducted at four degrees Celsius. The purified mitochondria can now be tested to determine the metabolic activity of the isolated mitochondria. An A TP luminescence assay can be performed using an A TP assay kit.

First, bring the kit reagents to room temperature. Next, prepare the HT P stock solution and place it on ice. The next step is to add five milliliters of substrate buffer to the lyophilized substrate solution.

Mix this gently and place in the dark. Then add 100 microliters of respiration buffer to each well of a 96 well plate that is opaque black. Add 10 microliters of the prepared mitochondria to wells below the A TP standards.

Each unique mitochondrial sample should be plated in triplicate plate three wells of respiration buffer alone as a negative control. Continue by adding 50 microliters of mammalian cell lysis solution to every well once loaded. Incubate the plate at 37 degrees Celsius for five minutes on an orbital shaker at 120 RPM.

During the incubation, prepare dilutions of the A TP standard ranging from one 10th millimolar to one 10th micromolar. Following the incubation, add 10 microliters of each of the A TP standards to corresponding wells indicated on the plate map. Next, add 50 microliters of the reconstituted substrate solution to each.

Well then incubate the plate again, as before for five minutes. Proceed by examining the plates at the plate reader. Open the software under.

Create a new item. Click on experiment. Then click on default protocol and click okay.

In the left column, select protocol. Then procedure Next, select delay, and set it to 10 minutes. Then okay, the entry.

Now select, read and choose luminescence from the dropdown menu. Adjust the other settings as follows. Read type endpoint integration time.

One second. Filter sets one emission hole optics position, top sensitivity 100, top probe vertical offset one millimeter. After making these setting changes, click okay.

Click on plate layout and set it up accordingly. Click okay. Now with everything set, click the read plate icon on the top row and click read.

When the spectrophotometer opens, place the plate into the tray with well A one in the upper left corner. Now a box displaying the temperature will open, click okay, then proceed with analysis of the data. Skeletal muscle samples weighing about 0.18 grams wet yielded about 24 billion mitochondria based on particle size counting.

Liver samples of the same size gave a few billion more. Mitochondria hemo cytometer readings were underestimated using the particle counter. A representative tracing was obtained, which shows that isolated mitochondria are localized under one peak with a mean diameter of about four tenths of a micron.

This is in agreement with previous reports using the bi acid assay. Mitochondrial protein was measured at a few milligrams per gram of tissue from skeletal muscle and liver. The product was viable and respiration competent.

Mitochondria isolated in a timeframe compatible for clinical and surgical therapeutic intervention. Transmission electron micrographs indicated that isolated mitochondria were all electron dents with damage to less than one in 10, 000, contamination was at less than one in a hundred thousand A TP assay. Results revealed that mitochondria were viable and that a TP content was around 11 or 15 nanomoles per milligram of mitochondrial protein.

Mitochondrial respiration analysis was conducted using a Clark type electrode and revealed that oxygen consumption was about 180 MLEs of oxygen per minute. For every milligram of mitochondrial protein while RCI values were each around 2.5 with a slightly higher liver score Once mastered, this technique can be done in less than 30 minutes if it is performed properly.I.