Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

The overarching aim of this procedure is to immuno label proteins of interest on the ER mitochondrial membrane with an antibody of your choice and analyze concurrently with mitochondrial function. This is accomplished by first collecting the tissue of interest such as the brain, spinal cord, or liver from a rat. The second step is to isolate the mitochondria from the tissue, in this case the spinal cord.

The spinal cord is homogenized with a dance. Homogenizer and other organelles and cell day debris are removed through a series of centrifugation steps until a crude mitochondrial palate is achieved myelin, a major contaminant of central nervous tissue is removed by mixing mitochondria with a solution of 12%opti prep and centrifusion once again. Next, the isolated mitochondria are labeled with a primary antibody of choice, followed by a secondary antibody conjugated to a Fluor.

The final step is to add fluorescent dyes to confirm mitochondrial identity or rather functional parameters. Ultimately, flow cytometry is used to show the relative abundance of mitochondrial alto membranes throughout normal physiology or disease. The main advantage of this technique over existing methods like microscopy is that it permits high throughput and rapid quantitation of thousands of individual mitochondria.

Animals used in this study were treated in strict accordance to a protocol approved by the C-R-C-H-U-M institutional Committee for the protection of animals, which follows national standards as outlined by the Canadian Council on Animal Care Air. To begin this procedure, place a whole intact spinal cord that was collected from a rat in a five milliliter glass homogenizer containing five volumes of homogenization buffer. Place the glass homogenizer on ice and homogenize the tissue by hand until no larger pieces of tissue remain, which will take approximately eight strokes.

Transfer the homogenate to two or three micro centrifuge tubes and centrifuge at 1, 300 times G for 10 minutes of four degrees Celsius us next pipette The S supernatant into a five milliliter ultracentrifuge tube. Add 750 microliters of homogenization buffer to the pellet and gently resuspend the pellet. Then centrifuge the suspension and resuspend the pellet.

Two more times to remove small debris, pull all the supernatants into the same five milliliter ultracentrifuge tube, and then place the tubes into an ultracentrifuge rotor. Equipped with swinging buckets, centrifuge the tubes at 17, 000 times G for 15 minutes of four degrees Celsius. When the centrifugation is complete, save the supernatant for further processing if the cytosolic fraction is of interest.

Next, resus suspend the pellet, which is the crude mitochondrial fraction in four milliliters of homogenization buffer containing 50 millimolar potassium chloride centrifuge. The resuspended pellet at 17, 000 times G for 15 minutes at four degrees Celsius in a swinging bucket rotor, then discard the supinate and gently resuspend the pellet in 800 microliters of homogenization buffer in a new five milliliter ultracentrifuge tube at exactly 800 microliters of the resuspended pellet. Next pipette 200 microliters of iod dal to the 800 microliters of the resuspended pellet to create a final concentration of 12%iod Dal.

Mix the contents of the tube gently but thoroughly with a P 1000 pipette and then centrifuge at 17, 000 times G for 15 minutes at four degrees Celsius in a swinging bucket rotor aspirate the layer of myelin at the top of the tube and discard the supinate carefully as the palette may be loose. Resus suspend the pellet in four milliliters of homogenization buffer. Then centrifuge your gain at 17, 000 times G for 15 minutes at four degrees Celsius in a swinging bucket rotor.

Discard the supinate and resus. Suspend the pellet in four milliliters of homogenization buffer. Then repeat the centrifugation again and remove the supine agent reus.

Bend the final pellet in 100 to 200 microliters of homogenization buffer and transfer to a 1.7 milliliter micro centrifuge tube. Determine the amount of protein in each sample using a standard protein quantitation assay such as bionic acid assay or BCA. When preparing the samples for quantitation, dilute the samples and the standard curve in 2%sodium sulfate to ensure adequate solubilization of mitochondria.

For each staining mix to be tested, pipette 25 micrograms of the isolated mitochondria into a 1.7 milliliter micro centri tube. Make sure to include an unstained sample in each experiment as well as an appropriate isotype control for each antibody tested centrifuge each sample at 17, 000 times G for two minutes at four degrees Celsius in a benchtop micro centrifuge. Then remove the supinate and resuspend the isolated mitochondria in 50 microliters of mitochondria buffer.

Supplemented with 10%fatty acid-free BSA incubate the solution for 15 minutes at four degrees Celsius, and then add 20 micrograms of the primary antibody per milliliter of solution. Incubate the isolated mitochondria with the primary antibody for 30 minutes at four degrees Celsius to wash out the unbound antibody, centrifuge each sample and discard the supine natant. Next, gently resuspend the pellet in 200 microliters of mitochondria buffer, and then repeat the centrifugation step, discard the supine natant and resuspend the pellet in 50 microliters of mitochondria buffer.

Add 0.5 micrograms of the secondary antibody per milliliter of sample. Then incubate the samples for 30 minutes at four degrees Celsius, protected from light. When the incubation is complete, centrifuge each sample and discard the supinate which contains the unbound secondary antibody.Reese.

Suspend the pellet in 200 microliters of mitochondria buffer and centrifuge the sample.Again. Then remove the supinate and Reese. Suspend the pellet in 500 microliters of mitochondria buffer to ensure that the observed events are in fact mitochondria.

Stain the sample with a mitochondria specific fluorescent dye such as MIT tracker green from in Vitrogen for 15 minutes in the dark. If no further staining is necessary to investigate other functional parameters, then transfer the labeled sample to a tube suitable for loading in a flow cytometer. Keep the samples on ice and proceed to a flow cytometer for acquisition to verify that the isolated mitochondria have an intact transmembrane potential stain the mitochondria with 100 nanomolar tetraethyl rod domine methyl ester for 15 minutes at room temperature in the dark as a control for staining with tetraethyl rod domine.

Methyl ester stain the isolated mitochondria with 100 nanomolar tetraethyl rod domine methyl ester in the presence of 100 micromolar carbonyl cyanide m chloro phenol HydroOne. This chemical is a mitochondrial uncouple and will depolarize the mitochondria to verify that the isolated mitochondria produce superoxide stain the mitochondria with an appropriate superoxide indicator such as mitoses socks red from in Vitrogen for 15 minutes at room temperature in the dark as a control for mitochondrial superoxide production. Stay in the isolated mitochondria with dye in the presence of 10 micromolar antimycin.

A antimycin A is an inhibitor of complex three of the respiratory chain and will augment mitochondrial superoxide production before the acquisition begins. Set up the instrument as described in the accompanying text protocol. Then mix the samples by gently tapping the tube, but do not vortex.

Begin collecting the initial events at a low pressure during gating. Make sure to gate on total population. Next, adjust the voltages of the histograms accordingly.

The peak of the unstained sample usually corresponds to the second decade. Then once the gates are established and samples are being processed, switch the pressure to high flow cytometry. Analysis of the spinal cord preparations identified that 94%of the events were positive for the mitochondria specific dye.

In this experiment, 30%of the mitochondria positive events were labeled with an antibody specific for MIT fusion two, which is a protein implicated in the fusion of the outer membrane of mitochondria. Analysis of the mitochondrial transmembrane potential using tetraethyl rumine methyl ester found that 95%of the isolated mitochondria have an intact transmembrane potential. This staining was shown to be specific as a significant decrease in staining was seen when performed in the presence of a mitochondrial unco functional mitochondria were found to produce a basal amount of SUP oxide as measured using a membrane permeable dye.

That becomes fluorescent P reaction with SUP oxide. Treatment of mitochondria with antimycin a results in an approximate 10%increase in superoxide production Once mastered. This technique can be done in six to seven hours if it is performed properly.