Qproteome Mitochondria Isolation Kit

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Publication protocol

Cell disruption and mitochondria extraction: Cells were disrupted using three approaches, mechanical disruption (Dounce Homogenizer), the proposed microscale cell shredder and a commercially available chemical-based cell membrane disruption kit (Qproteome® Mitochondria Isolation Kit), as detailed in the following. Dounce Homogenizer (Kimble® Tissue Grinder Comp, catalog no. 885300-0015) was utilized to mechanically disrupt cell membrane for mitochondria extraction. Cell disruption and mitochondria extraction were performed following a previously validated protocol53. Prior to mitochondrial extraction, the harvested cells were washed with pre-chilled phosphate-buffered saline (PBS, pH 7.4) and cell pellets were collected via trypsinization. Titrated concentrations (5–40 × 104 cells/mL) of cells were swelled by resuspending in the hypotonic Reticulocyte Standard Buffer (RSB) (10 mM NaCl, 1.5 mM MgCl2, 10 mM Tris-HCl, pH 7.5) and incubated at 37 °C for 10 min. The swelled cells (200 µL) were then transferred into the cylinder of the homogenizer and optimized number of strokes were applied to produce cell lysates. The optimized number of strokes were chosen based on the maximal mitochondrial extraction and total protein yield as shown in Supplementary Figure S1. In the investigation for the effect of tonicity in cell disruption, cells were resuspended in the isotonic buffer (PBS, pH 7.4) and lysed subsequently. Mitochondria were then separated from the cell lysates by differential centrifugation53. Unbroken cell, nuclei, and cell debris were removed by centrifuging the lysates twice at 1,000 g for 5 min. The supernatant was then centrifuged at 15,000 g for 15 min to obtain the mitochondrial pellet. The pelleted mitochondria were suspended in 1× mitochondrial storage (MS) buffer (10 mM Tris-HCl, pH 6.7, 10 mM KCl, 0.15 mM MgCl2, 1 mM PMSF, and 1 mM DTT) and used directly for the subsequent characterizations.
For the microscale cell shredder, cells were swelled by incubating with the RSB hypotonic buffer (37 °C, 10 min), or resuspended in the isotonic buffer (PBS, pH 7.4) and shredded by flowing (200 µL) through the designed microfluidic chip at the optimized (80 μL/min) volumetric rate. Sample collected from the outlet was then processed following the differential centrifugation steps exactly as above-mentioned and the extracted mitochondria were stored in the 1× MS buffer and analyzed without further processing.
Qproteome® Mitochondria Isolation Kit (Qiagen, catalog no. 37612) was used to chemically lyse the cells for mitochondrial extraction. The procedures for cell lysis and subsequent centrifugations were conducted following the manufacturer’s protocols. Firstly, fresh cell pellets were suspended in 200 µL of ice-cold Lysis Buffer (supplied by the Qiagen Kit), and incubated at 4 ˚C for 10 min. The cell solution was centrifuged at 1,000 g for 10 min. The precipitated cell pellets were then resuspended in 200 µL ice-cold Disruption Buffer (supplied by the Kit) and disrupted by repeatedly (10 times) passing through a narrow-gauge needle (22AWG, 120 µm inner diameter). Produced cell lysate was centrifuged at 1,000 g for 10 min to remove the large cell debris. The supernatant was then centrifuged at 6,000 g for 10 min to obtain the mitochondrial pallets. The mitochondrial pellets were washed with 200 µL Mitochondria Storage buffer (provided by the Kit) and finally isolated by centrifugation at 6,000 g for 20 min.
All the differential centrifugation steps were conducted at 4 °C using a high-speed refrigerated centrifuge (Neofuge 13 R, Heal Force).

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