PRODUCTS AND METHODS TO ISOLATE MITOCHONDRIA

Abstract

Filtration apparatuses, kits, and methods for rapid isolation of intact, viable mitochondria from tissues are described with mitochondria isolated by differential filtration through nylon mesh filters. Mitochondria can be isolated in less than 30 minutes using the filtration apparatuses, kits, and methods described.

Claims

1. A method for isolating a viable mitochondrion, the method comprising: providing a cell homogenate comprising a viable mitochondrion; passing the cell homogenate through a first filter having a pore-size of about 30 μm to about 50 μm; and subsequently passing the cell homogenate through a second filter having a pore-size of about 5 μm to about 20 μm to thereby form a filtrate; and collecting the filtrate, thereby isolating the viable mitochondrion.

2. The method of claim 1, wherein the method comprises passing the cell homogenate through a third filter after the first filter and before the second filter, wherein the third filter has a pore-size of about 15 μm to about 50 μm.

3. The method of claim 1, wherein the method comprises homogenizing a tissue in a solution comprising 300 mM sucrose; 10 mM K.sup.+HEPES, pH 7.2; and 1 mM K.sup.+EGTA, pH 8.0, to thereby provide the cell homogenate.

4. The method of claim 1, wherein the method comprises, prior to introducing the cell homogenate, wetting the second filter with a solution comprising 1 mg BSA in 1 mL of a solution comprising 300 mM sucrose; 10 mM K.sup.+HEPES, pH 7.2; and 1 mM K.sup.+EGTA, pH 8.0.

5. (canceled)

6. The method of claim 1, wherein the filtrate is centrifuged at 9000 rpm at 4° C. for five minutes.

7. The method of claim 1, wherein the cell homogenate is provided by homogenizing tissue in a sterile glass-grinding vessel.

8. A filtration apparatus comprising: a tubular body configured to be received in a centrifuge tube and comprising a lumen and first and second ends, each end comprising an opening; a first filter disposed and secured within the lumen, wherein the filter has a pore-size of about 30 μm to about 50 μm; and a second filter disposed and secured within the lumen adjacent to the first filter and having a pore-size of about 5 μm to about 20 μm.

9. The apparatus of claim 8, wherein the filtration apparatus comprises a third filter disposed and secured within the lumen adjacent to the first filter and the second filter and having a pore-size of about 15 μm to about 50 μm.

10. The apparatus of claim 8, wherein the apparatus is sterile.

11. The apparatus of claim 8, wherein the first filter comprises nylon, mylar, stainless steel, wire mesh, aluminum, synthetic mesh, spectra, Kevlar, plastic, or paper.

12. The apparatus of claim 8, wherein the first filter comprises pores with a size of about 40 μm.

13. The apparatus of claim 8, wherein the second filter comprises nylon, mylar, stainless steel, wire mesh, aluminum, synthetic mesh, spectra, Kevlar, plastic, or paper.

14. The apparatus of claim 8, wherein the second filter comprises pores with a size of about 10 μm.

15. The apparatus of claim 9, wherein the third filter comprises nylon, mylar, stainless steel, wire mesh, aluminum, synthetic mesh, spectra, Kevlar, plastic, or paper.

16. The apparatus of claim 9, wherein the third filter comprises pores with a size of about 20 μm.

17. The apparatus of claim 8, wherein the centrifuge tube is a 50 mL centrifuge tube.

18. A kit, the kit comprising a filtration apparatus comprising: a tubular body configured to be received in a centrifuge tube and comprising a lumen and first and second ends, each end comprising an opening; a first filter disposed and secured within the lumen, wherein the filter has a pore-size of about 30 μm to about 50 μm; and a second filter disposed and secured within the lumen adjacent to the first filter and having a pore-size of about 5 μm to about 20 μm.

19. The kit of claim 18, wherein the kit further comprises: a first solution comprising 300 mM sucrose; 10 mM K.sup.+HEPES, pH 7.2; and 1 mM K.sup.+EGTA, pH 8.0; a second solution comprising 2 mg Subtilisin A per 1 mL of the first solution; a third solution comprising 10 mg BSA per 1 mL of the first solution; a fourth solution comprising 1 mg BSA per 1 mL of the first solution; a 50 mL centrifuge tube; and a 1.5 mL microcentrifuge tube.

20. The kit of claim 19, wherein the 50 mL centrifuge tube and the 1.5 mL microcentrifuge tube are sterile.

21. A method for isolating a viable mitochondrion, the method comprising: providing a cell homogenate comprising a viable mitochondrion; providing the filtration apparatus of claim 8; passing the cell homogenate through the apparatus to thereby form a filtrate comprising a viable mitochondrion; and collecting the filtrate, thereby isolating the viable mitochondrion.

Description

DESCRIPTION OF DRAWINGS

[0012] FIG. 1 is a schematic diagram showing an exemplary filtration apparatus.

[0013] FIG. 2 shows a scheme for isolating mitochondria using tissue dissociation and differential filtration in a total procedure time of less than 30 minutes.

[0014] FIG. 3A is a bar graph showing hemocytometer and particle size counter mitochondria number isolated from 0.18±0.04 g tissue (wet weight) for skeletal muscle and liver.

[0015] FIG. 3B is a line graph showing mitochondrial size distribution as detected by particle size counter.

[0016] FIG. 3C is a bar graph showing mitochondrial protein (mg/g) tissue wet weight for skeletal muscle and liver.

[0017] FIG. 3D is a transmission electron microscopy image of isolated mitochondria. Scale bar is 100 nm. Arrows indicate possible contamination by non-mitochondrial particles and damaged mitochondria.

[0018] FIG. 4A is a photomicrograph of isolated mitochondria under phase contrast illumination. Scale bar is 25 μm.

[0019] FIGS. 4B and 4C are photomicrographs of isolated mitochondria under fluorescence illumination, with mitochondria labeled with MitoTracker Red CMXRos. Scale bars are 25 μm (FIG. 4B) and 5 μm (FIG. 4C).

[0020] FIG. 4D is a bar graph depicting ATP content nmol/mg mitochondrial protein as determined by ATP assay.

[0021] FIG. 4E is a bar graph showing Respiratory Control Index (RCI) (state 3/state 4) as determined by Clark electrode.

[0022] FIG. 5 is a table showing a plate map for ATP assay.

DETAILED DESCRIPTION

[0023] Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation typically require 60 to 100 minutes to complete. Described herein are filtration apparatuses, kits, and methods for rapid isolation of mitochondria from tissues. Certain methods described herein employ a tissue dissociator and differential filtration. In this method, manual homogenization can be replaced with the tissue dissociator's standardized homogenization cycle, which allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the cell homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 minutes. A typical isolation using the filtration apparatuses, kits, and methods described herein can yield approximately 2×10.sup.10 viable and respiration-competent mitochondria from 0.18±0.04 g (wet weight) of tissue sample.

[0024] Filtration apparatuses described herein can be used to rapidly isolate intact, viable mitochondria in 30 minutes or less, e.g., 28 minutes, 25 minutes, or 20 minutes or less. Employing differential filtration in place of standard differential centrifugation in methods of isolating mitochondria significantly reduces procedure time and subjects mitochondria to less mechanical stress than using standard differential centrifugation protocols. For example, protocols incorporating several centrifugation steps can take 60 minutes to 100 minutes to isolate mitochondria (Frezza et al., Nat Protoc 2:287-295, 2007; Wieckowski et al., Nat Protoc 4:1582-1590, doi: 10.1038/nprot.2009.151, 2009; Gostimskaya et al., J Vis Exp (43), pii:2202, doi: 10.3791/2202, 2010; Gross et al., Anal Biochem 418:213-223, doi: 10.1016/j.ab.2011.07.017, 2011; Masuzawa et al., Amer J Physiol Heart Circ Physiol 304:H966-H982, doi: 10.1152/ajpheart.00883.2012, 2013). Another advantage of the present filtration apparatuses, kits, and isolation methods is that tissue homogenization is standardized. A tissue dissociator provides a standardized cycle and yields consistent and reproducible results. This is in contrast to manual homogenization that is subject to user variability and inconsistency. The isolation time frame provided by the present methods is compatible for clinical and surgical therapeutic intervention (Masuzawa et al., Amer J Physiol Heart Circ Physiol 304:H966-H982, doi: 10.1152/ajpheart.00883.2012, 2013; McCully et al., Amer J Physiol Heart Circ Physiol 296:H94-H105, doi: 10.1152/ajpheart.00567.2008, 2009).

Apparatuses

[0025] Filtration apparatuses described herein feature a body (e.g., a tubular body) configured to house multiple filters, which are further described below and represented in FIG. 1. Body (10) can be shaped to fit into a centrifuge tube (90). Body (10) and centrifuge tube (90) can be of any size. Body (10) has first and second ends (20) and (80), each end having an opening (30) and (70). The body has a lumen (40), such that a sample can be placed into the body at one end, travel through lumen (40), and be retrieved at the opposite end, after the sample progresses through at least two filters disposed within the body, e.g., by force of gravity, capillary action, and/or centrifugation. Skilled practitioners will appreciate that one or both ends (20 and/or 80), while each has an opening, can in some embodiments be reversibly capped, e.g., to preserve sterility and/or provide an area to collect filtrate. Typically, the body will have a roughly circular cross-section along the length of the body. However, skilled practitioners will appreciate that the cross-section of the body can be of any shape desired, e.g., oval, square, rectangular, triangular, etc. In some embodiments, the cross-section is roughly circular so that the body can be received within a commercially-available centrifuge tube (90), e.g., a 50 mL centrifuge tube. Skilled practitioners will appreciate that tubular body (10) can be constructed from any art-known material, e.g., polypropylene or polystyrene. One end of the body, e.g., end (80), can have a tapered configuration (shown in FIG. 1) to facilitate insertion of the body into a centrifuge tube and/or to aid in retaining the filter(s) within the body.

[0026] The apparatuses include a first filter (50) disposed and secured within the lumen, wherein the filter has a pore-size of about 30 μm to about 50 μm, e.g., about 30 μm, 33 μm, 35 μm, 38 μm, 40 μm, 42 μm, 45 μm, 48 μm, or about 50 μm. The first filter can be constructed from any art-known filter material, e.g., nylon, mylar, stainless steel, wire mesh, aluminum, synthetic mesh, spectra, Kevlar, plastic, paper, or any combination thereof. The apparatuses also include a second filter (60) disposed and secured within the lumen adjacent to the first filter and have a pore-size of about 5 μm to about 20 μm, e.g., about 5 μm, 6 μm, 8 μm, 10 μm, 12 μm, 15 μm, 18 μm, or about 20 μm. The second filter can be constructed from any art-known filter material, e.g., nylon, mylar, stainless steel, wire mesh, aluminum, synthetic mesh, spectra, Kevlar, plastic, paper, or any combination thereof. The first and second filters can be situated within the body such that they contact each other or are spaced apart some distance apart. For example, the first and second filters can be disposed within the body at a distance apart of at least or about 0.5 mm, e.g., at least or about 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 8 mm, 1 cm, 2 cm, 5 cm, or at least or about 10 cm, depending on the intended use and/or the length of the body.

[0027] The apparatuses can optionally include a third filter disposed and secured within the lumen adjacent to the first filter and the second filter and have a pore-size of about 15 μm to about 50 μm, e.g., about 15 μm, 18 μm, 20 μm, 22 μm, 25 μm, 28 μm, 30 μm, 33 μm, 35 μm, 38 μm, 40 μm, 42 μm, 45 μm, 48 μm, or about 50 μ. The third filter can be constructed from any art-known filter material, e.g., nylon, mylar, stainless steel, wire mesh, aluminum, synthetic mesh, spectra, Kevlar, plastic, paper, or any combination thereof. The third filter can be disposed between the first and second filters. In some embodiments, the third filter is disposed between the first filter and first opening, i.e., closer to the first opening than the first filter. In other embodiments, the third filter is disposed between the second filter and the second opening, i.e., closer to the second opening than the second filter. The third filter can be disposed such that it contacts the first filter and/or the second filter, or the three filters can be evenly spaced apart, or unevenly spaced apart. For example, when the third filter is disposed between the first and second filters, the first and third filters can be in contact with each other, or at least or about 0.5 mm apart, e.g., at least or about 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 8 mm, 1 cm, 2 cm, 5 cm, or at least or about 10 cm apart; and the third and second filters can be in contact with each other, or at least or about 0.5 mm apart, e.g., at least or about 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 8 mm, 1 cm, 2 cm, 5 cm, or at least or about 10 cm apart.

[0028] Skilled practitioners will appreciate that further filters (e.g., a fourth filter, fifth filter, sixth filter, etc.) may in some instances be added, depending on the intended use. In some embodiments, the apparatus is sterile. Skilled practitioners will appreciate that filters can be secured within the body using any art-known method, e.g., using adhesive, a pressure fit, and/or configuring the lumen walls in a way that causes the filters to be retained in the lumen (e.g., by designing lumen walls to have ridges, grooves, or other retentive elements).

Kits

[0029] The present disclosure also provides kits featuring the filtration apparatuses described herein to isolate viable mitochondria. Such kits include at least one, e.g., two, three, five, or ten, filtration apparatus described above. The kits can further include one or more solutions useful for performing the mitochondria isolation methods described herein. For example, a kit may include a first solution comprising 300 mM sucrose; 10 mM K.sup.+HEPES, pH 7.2; and 1 mM K.sup.+EGTA, pH 8.0. Alternatively or in addition, the kit may include a second solution comprising 2 mg Subtilisin A per 1 mL of the first solution. Alternatively or in addition, the kit may include a third solution comprising 10 mg BSA per 1 mL of the first solution. Alternatively or in addition, the kit may include solutions comprising inactive human serum albumin or acetylated human serum albumin. Alternatively or in addition, the kit may include a fourth solution comprising 1 mg BSA per 1 mL of the first solution.

[0030] In some instances, the kit may include a 50 mL centrifuge tube, into which the filtration apparatus can be fitted. Alternatively or in addition, the kit can include a 1.5 mL microcentrifuge tube. In some embodiments, the 50 mL centrifuge tube and the 1.5 mL microcentrifuge tube are sterile.

Methods of Isolating Mitochondria

[0031] In an exemplary method, intact, viable mitochondria are isolated from tissue, e.g., mammalian tissue, e.g., mammalian tissue from a tissue biopsy. For example, tissue from a mammal can be minced, e.g., with a scalpel, and homogenized in a sterile glass-grinding vessel (Thomas, Philadelphia, PA) with a motor-driven pestle for 5 to 10 seconds at 4° C. in a first solution containing 300 mM sucrose; 10 mM K.sup.+HEPES, pH 7.2; and 1 mM K.sup.+EGTA, pH 8.0. A solution containing 2 mg Subtilisin A per 1 mL of the first solution is then added to the homogenate and incubated on ice for 10 minutes.

[0032] After incubation on ice, the cell homogenate is introduced to a sterile filtration apparatus that is positioned relatively upright, as described herein. In some embodiments, a volume of the cell homogenate, e.g., about 100 μL, 200 μL, 300 μL, 400 μL, 500 μL, 600 μL, 700 μL, 800 μL, 900 μL, 1 mL, 1.5 mL, 2 mL, 2.5 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 45 mL, or about 50 mL, is introduced into the opening at the first end such that the cell homogenate contacts the first filter prior to contacting the second filter, and a filtrate is collected after passing through both filters, e.g., by gravity or by centrifugation, in a tube, e.g., a centrifuge tube, a vial, a microcentrifuge tube, or a test tube, to isolate the intact, viable, respiration-competent mitochondria. Alternatively or in addition, the filtration apparatus can have a cap on the second end that is able to collect the filtrate, and the cap can be uncapped or unscrewed to collect the filtrate after the filtrate has flowed through the first filter, second filter, and, if present, third filter, by gravity or centrifugation. In some embodiments, a volume of the cell homogenate can be passed through a filter with a pore-size of about 30 μm to about 50 μm, e.g., about 33 μm, 35 μm, 38 μm, 40 μm, 42 μm, 45 μm, or about 48 μm, and optionally, the filtrate passed through another filter with a pore-size of about 15 μm to about 50 μm, e.g., about 18 μm, 20 μm, 22 μm, 25 μm, 28 μm, 30 μm, 33 μm, 35 μm, 38 μm, 40 μm, 42 μm, 45 μm, or about 48 μm, before being passed through a filter with a pore-size of about 5 μm to about 20 μm, e.g., about 6 μm, 8 μm, 10 μm, 12 μm, 15 μm, or about 18 μm.

[0033] Prior to introducing the cell homogenate to the filtration apparatus, the filter with a pore-size of about 5 μm to about 20 μm, e.g., about 6 μm, 8 μm, 10 μm, 12 μm, 15 μm, or about 18 μm, can be wetted with a solution comprising 1 mg BSA in 1 mL of a solution comprising 300 mM sucrose; 10 mM K.sup.+HEPES, pH 7.2; and 1 mM K.sup.+EGTA, pH 8.0. While not always required in the methods described herein, skilled practitioners will appreciate that filtrate collection can be facilitated by centrifuging the apparatus, e.g., at 1×g for three minutes. Skilled practitioners will appreciate that mitochondria can be concentrated by centrifuging the filtrate at 9000 rpm at 4° C. for five minutes.

EXAMPLES

[0034] Several general protocols are described below, which may be used in any of the methods described herein and do not limit the scope of the invention described in the claims.

Example 1: Stock Solutions

[0035] The following solutions were prepared to isolate intact, viable, respiration-competent mitochondria. To successfully isolate mitochondria using the present methods, all solutions and tissue samples should be kept on ice to preserve mitochondrial viability. Even when maintained on ice, isolated mitochondria will exhibit a decrease in functional activity over time (Olson et al., J Biol Chem 242:325-332, 1967). All solutions should be pre-prepared if possible.

[0036] 1 M K-HEPES Stock Solution (adjust pH to 7.2 with KOH).

[0037] 0.5 M K-EGTA Stock Solution (adjust pH to 8.0 with KOH).

[0038] 1 M KH.sub.2PO.sub.4 Stock Solution.

[0039] 1 M MgCl.sub.2 Stock Solution.

[0040] Homogenizing Buffer (pH 7.2): 300 mM sucrose, 10 mM K-HEPES, and 1 mM K-EGTA. Stored at 4° C.

[0041] Respiration Buffer: 250 mM sucrose, 2 mM KH.sub.2PO.sub.4, 10 mM MgCl.sub.2, 20 mM K-HEPES Buffer (pH 7.2), and 0.5 mM K-EGTA (pH 8.0). Stored at 4° C.

[0042] 10×PBS Stock Solution: 80 g of NaCl, 2 g of KCl, 14.4 g of Na.sub.2HPO.sub.4, and 2.4 g of KH.sub.2PO.sub.4 were dissolved in 1 L double distilled H.sub.2O (pH 7.4).

[0043] 1×PBS was prepared by pipetting 100 mL 10×PBS into 1 L double distilled H.sub.2O.

[0044] Subtilisin A Stock was prepared by weighing out 4 mg of Subtilisin A into a 1.5 mL microfuge tube. Stored at −20° C. until use.

[0045] BSA Stock was prepared by weighing out 20 mg of BSA into a 1.5 mL microfuge tube. Stored at −20° C. until use.

Example 2: Mitochondrial Isolation

[0046] A figure outlining the procedural steps in the isolation of mitochondria using tissue dissociation and differential filtration is shown in FIG. 2. Two, 6 mm biopsy sample punches were transferred to 5 mL of Homogenizing Buffer in a dissociation C tube and the samples were homogenized using the tissue dissociator's 1 minute homogenization program (A). Subtilisin A stock solution (250 μL ) was added to the homogenate in the dissociation C tube and incubated on ice for 10 minutes (B). The homogenate was filtered through a pre-wetted 40 μm mesh filter in a 50 mL conical centrifuge tube on ice and then 250 μL of BSA stock solution was added to the filtrate (C). The filtrate was re-filtered through a new pre-wetted 40 μm mesh filter in a 50 mL conical centrifuge on ice (D). The filtrate was re-filtered through a new pre wetted 10 μm mesh filter in a 50 mL conical centrifuge tube on ice (E). The filtrate was transferred to 1.5 mL microfuge tubes and centrifuged at 9000×g for 10 minutes at 4° C. (F). The supernatant was removed, and pellets containing mitochondria were re-suspended, and combined in 1 mL of Respiration Buffer (G).

[0047] Immediately prior to isolation, Subtilisin A was dissolved in 1 mL of Homogenizing Buffer. Immediately prior to isolation, BSA was dissolved in 1 mL of Homogenizing Buffer. Two fresh tissue samples were collected using a 6 mm biopsy sample punch and stored in 1×PBS in a 50 mL conical centrifuge tube on ice. The two 6 mm punches of tissue were transferred to a dissociation C tube containing 5 mL of ice cold Homogenizing Buffer. The tissue was homogenized by fitting the dissociation C tube on the tissue dissociator and selecting the pre-set mitochondrial isolation cycle (60 second homogenization).

[0048] The dissociation C tube was removed to an ice-bucket. Subtilisin A Stock Solution (250 μL) was added to the homogenate, mixed by inversion, and the homogenate was incubated on ice for ten minutes. A 40 μm mesh filter was placed onto a 50 mL conical centrifuge tube on ice and the filter was pre-wet with Homogenizing Buffer, and the homogenate was filtered into the 50 mL conical centrifuge tube on ice.

[0049] Freshly prepared BSA Stock Solution (250 μL) was added to the filtrate and mixed by inversion. (This step was omitted if mitochondrial protein determination was required.) A 40 μm mesh filter was placed onto a 50 mL conical centrifuge tube on ice and the filter was pre-wet with Homogenizing Buffer, and the homogenate was filtered into the 50 mL conical centrifuge tube on ice. A 10 μ.m filter was placed onto the 50 mL conical centrifuge tube on ice, and the filter was pre-wetted with Homogenizing Buffer, and the homogenate was filtered into the 50 mL conical centrifuge tube on ice. The filtrate was transferred to two pre-chilled 1.5 mL microfuge tubes and centrifuge at 9000×g for 10 minutes at 4° C. The supernatant was removed, and the pellets were re-suspended and combined in 1 mL of ice-cold Respiration Buffer.

Example 3: ATP Assay

[0050] To determine the metabolic activity of isolated mitochondria, an ATP luminescence assay was performed using an ATP assay kit. The protocol, reagents and standards were supplied in the assay kit. A summary of the procedure is described below.

[0051] Kit reagents were equilibrated to room temperature. 10 mM ATP Stock Solution was prepared by dissolving lyophilized ATP pellet in 1,170 μL of double distilled water. ATP standard Stock Solution and prepared mitochondrial samples were stored on ice.

[0052] Substrate Buffer solution (5 mL) was added to a vial of lyophilized substrate solution, mixed gently, and placed in the dark. Respiration Buffer (100 μL) was added to all wells of a black, opaque bottom, 96 well plate. Mitochondria from the prepared samples (10 μL) were added to each well of the 96 well plate. Samples were plated in triplicate, and a row for standards and three wells for the negative control (Respiration Buffer) were included. Mammalian cell lysis solution (50 μL) was added to all wells, including standards and controls. The 96 well plate was incubated at 37° C. for 5 minutes on an orbital shaker at 125 rpm. During the incubation, ATP standards were prepared in concentrations of 0.1 mM, 0.05 mM, 0.01 mM, 0.005 mM, 0.001 mM, and 0.0001 mM ATP from the 10 mM ATP Stock Solution and stored on ice. Following the incubation, 10 μL of ATP standards were add to corresponding wells as indicated on the plate map (FIG. 5). This plate map illustrates how to set up standards (A1-Al2), mitochondria samples (B1-C6), and negative controls (C7-C9) for the ATP assay. During the assay, 100 μL of Respiration Buffer, 50 μL of mammalian cell lysis solution, and 50 μL of reconstituted substrate solution are added to all wells (A1-C9). The reconstituted substrate solution (50 μL) was added to each well, and the 96 well plate was incubated at 37° C. on the orbital shaker for 5 minutes at 125 rpm.

[0053] The plate was read with a spectrophotometer controlled by Open GenS 1.11 software. Higher values correlate with increased ATP levels and higher metabolic activity.

Example 4: Representative Results

[0054] Tissue samples were obtained using a 6 mm biopsy punch. Tissue weight was 0.18±0.04 g (wet weight). The number of mitochondria isolated as determined by particle size counting was 2.4×10.sup.10±0.1×10.sup.10 mitochondria for skeletal muscle and 2.75×10.sup.10±0.1×10.sup.10 mitochondria for liver preparations (FIG. 3A). To allow for comparison, mitochondrial number was also determined by hemocytometer. Mitochondrial numbers were underestimated as determined by hemocytometer as 0.11×10.sup.10±0.04×10.sup.10 mitochondria for skeletal muscle and 0.34×10.sup.10±0.09×10.sup.10 mitochondria for liver preparations (FIG. 3A). Mitochondrial diameter as determined by size based particle counter is shown in FIG. 3B. The representative tracing shows the isolated mitochondria are localized under one peak with mean diameter of 0.38±0.17 μm in agreement with previous reports (Hogeboom et al., J Biol Chem 172:619-635, 1948).

[0055] Mitochondrial protein/g (wet weight) starting tissue, as determined by Bicinchoninic Acid (BCA) assay, was 4.8±2.9 mg/g (wet weight) and 7.3±3.5 mg/g (wet weight) for skeletal muscle and liver samples respectively (FIG. 3C).

[0056] Mitochondrial purity was determined by transmission electron microscopy and is shown in FIG. 3D. Mitochondria are shown to be electron dense with less than 0.01% being fractured or damaged. Contamination by non-mitochondrial particles is less than 0.001%.

[0057] Mitochondrial viability was determined by MitoTracker Red as previously described (Masuzawa et al., Amer J Physiol Heart Circ Physiol 304:H966-H982, doi: 10.1152/ajpheart.00883.2012, 2013; McCully et al., Amer J Physiol Heart Circ Physiol 296:H94-H105, doi: 10.1152/ajpheart.00567.2008, 2009). The present methods produce isolated mitochondria that maintain membrane potential (FIGS. 4A-C). These images indicate that mitochondria maintained membrane potential. Arrows indicate mitochondria lacking membrane potential or debris (FIG. 4A).

[0058] ATP was determined using a luminescent assay kit. A plate map for the ATP assay is shown in FIG. 5. ATP standards were plated in duplicate. Mitochondrial samples and negative controls were plated in triplicate. ATP content was 10.67±4.38 nmol/mg mitochondrial protein and 14.83±4.36 nmol/mg mitochondrial protein for skeletal muscle and liver samples, respectively (FIG. 4D).

[0059] Mitochondrial respiration was assessed using a Clark type electrode as previously described (Masuzawa et al., Amer J Physiol Heart Circ Physiol 304:H966-H982, doi: 10.1152/ajpheart.00883.2012, 2013; McCully et al., Amer J Physiol Heart Circ Physiol 296:H94-H105, doi: 10.1152/ajpheart.00567.2008, 2009). Mitochondrial oxygen consumption rate was 178±17 nM 02/min/mg mitochondrial protein for skeletal muscle and 176±23 nM O.sub.2/min/mg mitochondrial protein for liver preparations. Respiratory control index (RCI) values were 2.45±0.34 and 2.67±0.17 for skeletal muscle and liver sample preparations, respectively (FIG. 4E). These results are similar to those reported in previous studies using manual homogenization and differential centrifugation to isolate mitochondria (Masuzawa et al., Amer J Physiol Heart Circ Physiol 304:H966-H982, doi: 10.1152/ajpheart.00883.2012, 2013; McCully et al., Amer J Physiol Heart Circ Physiol 296:H94-H105, doi: 10.1152/ajpheart.00567.2008, 2009).

OTHER EMBODIMENTS

[0060] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.