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"Materials\r\n\r\n\r\n\tRecombinant mouse Bcl-xL (R&amp;amp;D Systems Catalog # 878-BC)\r\n\tCaspase-8-cleaved recombinant mouse BID (R&amp;amp;D Systems Catalog # 883-M8)\r\n\tCrude or enriched mouse liver mitochondria (See Preparation of Mitochondria from Mouse Liver)\r\n\tDilution Buffer: 25 mM HEPES-KOH (pH 7.4), 0.1 M KCl, 10% Glycerol, 1 mg/mL fatty acid free BSA* (Sigma Catalog # A6003)\r\n\tMitochondria Buffer: 125 mM KCl, 0.5 mM MgCl2, 3.0 mM Succinic acid, 3.0 mM Glutamic acid, 10 mM HEPES-KOH (pH 7.4), 1 mg/mL BSA*, containing 25 \u00B5g/mL Leupeptin, 25 \u00B5g/mL Pepstatin, 3 \u00B5g/mL Aprotinin, 100 \u00B5M PMSF, and 10 \u00B5M Boc-Asp-FMK caspase inhibitor\r\n\r\n\r\n*Note: Protease inhibitors and BSA should be added to the buffer immediately prior to use.\r\nBSA stock solution should be prepared at 100 mg/mL.\r\n\r\nMethods\r\n\r\nNote: All buffers, proteins, and tubes should be kept on ice until indicated.&amp;nbsp; Assay volumes are 75 \u00B5L and are combined in 0.5 mL Eppendorf tubes. The table below Step 5 summarizes the reactions that should be set up in Steps 1-6.\r\n\r\n\r\n\tPrepare a stock solution of Caspase-8-cleaved recombinant mouse BID (MW: 22 kDa) in Dilution Buffer at 9.0 \u00B5g/mL. The final concentration will be 54 nM.\r\n\tPrepare dilutions of recombinant mouse Bcl-xL (MW: 24.7 kDa) in Dilution Buffer at concentrations of 9000, 3000, 900, 300, 90, 30, 9, and 3 nM. The final concentration range will be 3000 to 1 nM in a total reaction volume of 75 \u00B5L.\r\n\tAliquot 25 \u00B5L of each of the Bcl-xL dilutions to a series of tubes. Add 10 \u00B5L of the Caspase-8-cleaved recombinant mouse BID stock solution to each tube and gently mix. Incubate for 60 minutes at room temperature. Include one sample without recombinant mouse Bcl-xL (cleaved BID control).\r\n\tAdd 12 \u00B5L of mitochondria (approximately 25-30 \u00B5g) and 28 \u00B5L of Mitochondria Buffer containing protease inhibitors and BSA to each tube.\r\n\tTwo control samples must be run for each assay to determine the total amount of Cytochrome c that can be released from the mitochondria and the amount of spontaneously released Cytochrome c. Set up two samples containing only mitochondria and the appropriate buffers that have not been treated with any test proteins.\r\n\tCap the tubes and gently mix the contents for 5-10 seconds. Incubate in a 30 \u00B0C water bath for 30 minutes.\r\n\t\r\n\t\t\r\n\t\t\t\r\n\t\t\t\tInitial Bcl-xL Conc.\r\n\t\t\t\tBcl-xL\r\n\t\t\t\tForm. \r\n\t\t\t\tCleaved \r\n\t\t\t\tDil. \r\n\t\t\t\tMito. \r\n\t\t\t\tMito.\r\n\t\t\t\tFinal \r\n\t\t\t\tFinal \r\n\t\t\t\r\n\t\t\t\r\n\t\t\t\t&amp;nbsp;\r\n\t\t\t\tVol.\r\n\t\t\t\tBuffer\r\n\t\t\t\tBID\r\n\t\t\t\tBuffer\r\n\t\t\t\tVol.\r\n\t\t\t\tBuffer&amp;nbsp;\r\n\t\t\t\tVol.\r\n\t\t\t\tConc.\r\n\t\t\t\r\n\t\t\t\r\n\t\t\t\t(nM)\r\n\t\t\t\t(uL)\r\n\t\t\t\t(uL)\r\n\t\t\t\t(uL)\r\n\t\t\t\t(uL)\r\n\t\t\t\t(uL)\r\n\t\t\t\t(uL)\r\n\t\t\t\t(uL)\r\n\t\t\t\t(nM)\r\n\t\t\t\r\n\t\t\t\r\n\t\t\t\t9000\r\n\t\t\t\t25\r\n\t\t\t\t-\r\n\t\t\t\t10\r\n\t\t\t\t-\r\n\t\t\t\t12\r\n\t\t\t\t28\r\n\t\t\t\t75\r\n\t\t\t\t3000\r\n\t\t\t\r\n\t\t\t\r\n\t\t\t\t3000\r\n\t\t\t\t25\r\n\t\t\t\t-\r\n\t\t\t\t10\r\n\t\t\t\t-\r\n\t\t\t\t12\r\n\t\t\t\t28\r\n\t\t\t\t75\r\n\t\t\t\t1000\r\n\t\t\t\r\n\t\t\t\r\n\t\t\t\t900\r\n\t\t\t\t25\r\n\t\t\t\t-\r\n\t\t\t\t10\r\n\t\t\t\t-\r\n\t\t\t\t12\r\n\t\t\t\t28\r\n\t\t\t\t75\r\n\t\t\t\t300\r\n\t\t\t\r\n\t\t\t\r\n\t\t\t\t300\r\n\t\t\t\t25\r\n\t\t\t\t-\r\n\t\t\t\t10\r\n\t\t\t\t-\r\n\t\t\t\t12\r\n\t\t\t\t28\r\n\t\t\t\t75\r\n\t\t\t\t100\r\n\t\t\t\r\n\t\t\t\r\n\t\t\t\t90\r\n\t\t\t\t25\r\n\t\t\t\t-\r\n\t\t\t\t10\r\n\t\t\t\t-\r\n\t\t\t\t12\r\n\t\t\t\t28\r\n\t\t\t\t75\r\n\t\t\t\t30\r\n\t\t\t\r\n\t\t\t\r\n\t\t\t\t30\r\n\t\t\t\t25\r\n\t\t\t\t-\r\n\t\t\t\t10\r\n\t\t\t\t-\r\n\t\t\t\t12\r\n\t\t\t\t28\r\n\t\t\t\t75\r\n\t\t\t\t10\r\n\t\t\t\r\n\t\t\t\r\n\t\t\t\t9\r\n\t\t\t\t25\r\n\t\t\t\t-\r\n\t\t\t\t10\r\n\t\t\t\t-\r\n\t\t\t\t12\r\n\t\t\t\t28\r\n\t\t\t\t75\r\n\t\t\t\t3\r\n\t\t\t\r\n\t\t\t\r\n\t\t\t\t3\r\n\t\t\t\t25\r\n\t\t\t\t-\r\n\t\t\t\t10\r\n\t\t\t\t-\r\n\t\t\t\t12\r\n\t\t\t\t28\r\n\t\t\t\t75\r\n\t\t\t\t1\r\n\t\t\t\r\n\t\t\t\r\n\t\t\t\t&amp;nbsp;\r\n\t\t\t\t&amp;nbsp;\r\n\t\t\t\t&amp;nbsp;\r\n\t\t\t\t&amp;nbsp;\r\n\t\t\t\t&amp;nbsp;\r\n\t\t\t\t&amp;nbsp;\r\n\t\t\t\t&amp;nbsp;\r\n\t\t\t\t&amp;nbsp;\r\n\t\t\t\t&amp;nbsp;\r\n\t\t\t\r\n\t\t\t\r\n\t\t\t\tControls\r\n\t\t\t\t&amp;nbsp;\r\n\t\t\t\t&amp;nbsp;\r\n\t\t\t\t&amp;nbsp;\r\n\t\t\t\t&amp;nbsp;\r\n\t\t\t\t&amp;nbsp;\r\n\t\t\t\t&amp;nbsp;\r\n\t\t\t\t&amp;nbsp;\r\n\t\t\t\t&amp;nbsp;\r\n\t\t\t\r\n\t\t\t\r\n\t\t\t\tCleaved BID only\r\n\t\t\t\t-\r\n\t\t\t\t25\r\n\t\t\t\t10\r\n\t\t\t\t-\r\n\t\t\t\t12\r\n\t\t\t\t28\r\n\t\t\t\t75\r\n\t\t\t\t0\r\n\t\t\t\r\n\t\t\t\r\n\t\t\t\tSpontaneous Release\r\n\t\t\t\t-\r\n\t\t\t\t25\r\n\t\t\t\t-\r\n\t\t\t\t10\r\n\t\t\t\t12\r\n\t\t\t\t28\r\n\t\t\t\t75\r\n\t\t\t\t0\r\n\t\t\t\r\n\t\t\t\r\n\t\t\t\tTotal Cytochrome c\r\n\t\t\t\t-\r\n\t\t\t\t25\r\n\t\t\t\t-\r\n\t\t\t\t10\r\n\t\t\t\t12\r\n\t\t\t\t28\r\n\t\t\t\t75\r\n\t\t\t\t0\r\n\t\t\t\r\n\t\t\r\n\t\r\n\t\r\n\tTotal Cytochrome c in the assay should be determined by freezing the entire 75 \u00B5L reaction mix immediately after incubation at 30 \u00B0C.\r\n\tCentrifuge the remaining samples at 16,000 x g for 5 minutes at 2-8 \u00B0C. Remove and transfer a 50 \u00B5L aliquot of the supernatant to a new chilled tube. Samples may be analyzed immediately or stored at -20 \u00B0C in a manual defrost freezer.\r\n\tTotal Cytochrome c in the assay should be determined by freezing the entire 75 \u00B5L reaction mix immediately after incubation at 30 \u00B0C.\r\n\tCentrifuge the remaining samples at 16,000 x g for 5 minutes at 2-8 \u00B0C. Remove and transfer a 50 \u00B5L aliquot of the supernatant to a new chilled tube. Samples may be analyzed immediately or stored at -20 \u00B0C in a manual defrost freezer.\r\n\tMeasure the levels of Cytochrome c in these samples using the mouse/rat Cytochrome c Quantikine\u00AE ELISA Kit (Catalog # MCTC0). See the Preparation of Samples for the Cytochrome c ELISA and additional instructions in the Cytochrome c Quantikine ELISA Kit product insert (Catalog # MCTC0).\r\n\r\n\r\nQuantikine is a registered trademark of R&amp;amp;D Systems.\r\n"^^<http://www.w3.org/2001/XMLSchema#string>
<https://www.rndsystems.com/protocol-types/apoptosis-assays>