PBI-Shredder HRR- Set: preparation of tissue homogenates
Contents
1. OROBOROS INSTRUMENTS high resolution respirometry Auxiliary HRR Tools Mitochondrial Physiology Network 17 02 1 9 2013 2012 2013 OROBOROS PBI Shredder HRR Set preparation of PBI Shredder tissue homogenates mw HRR Set for diagnosis of mitochondrial respiratory function A j T A Ry Anna Draxl Andrea Eigentler Erich Gnaiger 10ROBOROS INSTRUMENTS Corp high resolution respirometry Schopfstr 18 A 6020 Innsbruck Austria Email erich gnaiger oroboros at www oroboros at Medical University of Innsbruck Department of Visceral Transplant and Thoracic Surgery D Swarovski Research Laboratory 6020 Innsbruck Austria http wiki oroboros at index php K Regio MitoCom_ Tyrol 9 Q Q p Introduction Materials and chemicals 1 Components of the PBI Shredder HRR Set 2 Other materials 3 Media Sample preparation Organ harvest Tissue preparation Determination of wet weight Ww Quick protocol Detailed protocol tissue homogenization shredding Removing the homogenate Experimental setup with the Oxygraph 2k Conclusions References Author contributions and publication versions Section NDUBPWNE COPONNAUA ARAR UWV UWUUWVUNNANY The PBI Shredder HRR Set is an auxiliary HRR Tool providing a standardized approach to prepare homogenates of various tissues with high reproducibility of mitochondrial yield and mitochondrial function In this guide to applications with high resolution2. respirometry HRR we refer to instruments oroboros at www oroboros at MiPNet17 02 PBI Shredder HRR Set 2 the PBI User Manual for safety information product use limitations and warranty information and to the Product Specification Sheet by Pressure BioSciences Inc PBI 1 Introduction Application of high resolution respirometry i with gently prepared tissue homogenates ILOa Offers a versatile tool to study mitochondrial function in small amounts of tissues The PBI Shredder SG3 Figure 1 is a low shear mechanical homogenization system designed to apply reproducible force to the tissue with three positions of the force setting lever This yields standardized rapid and safe disruption of cells with preservation of intact functional mitochondria The laboratory specific or even operator specific protocols for tissue homogenization are thus standardized providing reproducible and consistent results for quantitative and inter laboratory comparison The easy handling enables especially beginners to obtain reliable results Figure 1 The PBLShredder SG3 The PBI Shredder HRR Set includes with handle red and torque driver Shredder Tubes for ambient pressure white assembled with the force Processing without and with a metal insert to setting lever metal ready for disrupt tough cellular structures In our application primary applications with mouse and fish myocard and liver Shredder Tubes with and without met
3. s Ice 2 3 Media e BIOPS The relaxing and organ preservation solution BIOPS contains 10 mM Ca EGTA buffer 0 1 uM free calcium 20 mM imidazole 20 mM taurine 50 mM K MES 0 5 mM DTT 6 56 mM MgCl 5 77 mM ATP 15 mM phosphocreatine pH 7 1 MiPNet0O3 02 BIOPS can be stored frozen at 20 C e MiROS5 MiROSCr MiRO6 or MiRO6Cr MiPNet14 13 3 Sample preparation 3 1 Organ harvest Heart and liver are excised from the sacrificed animal and immediately separated into specific subsamples and added into Falcon tubes containing sufficient ice cold BIOPS 30 ml for the entire mouse heart and trout heart or respiration medium trout liver to cover the entire tissue sample Keep on ice and minimize transportation and storage time as far as possible 3 2 Tissue preparation Place the tissue sample into a small Petri dish with fresh ice cold BIOPS or respiration medium on a cooling plate The tissue should be completely covered with liquid OROBOROS INSTRUMENTS www oroboros at MiPNet17 02 PBI Shredder HRR Set 4 Heart Open the left ventricle of the heart by using the dissecting scissors and forceps Cut out muscle tissue and omit pericardium Place small muscle pieces into a 12 well plate with ice cold respiration medium 3 3 Determination of wet weight Wy Prepare tissue samples of about 4 mg Ww of mouse heart muscle and about 16 mg Wy of trout heart muscle or trout liver for two O2k Chambers half the Wy if o
4. Description Product lproduct image id PBl Shredder SG3 PBIl Shredder SG3 for tissue homogenate 52100 preparation heavy duty high torque SG3 driver with convertible handle SG3 base with 3 position force setting lever FSL battery charger and two lithium ion batteries The PBl Shredder SG3 is included in the PBI Shredder HRR Set Select 230 V or 120 V OROBOROS INSTRUMENTS world wide distributor Shredder Kit Box Shredder Kit Box box for storage and 52101 PBI Shredder SG3 shipping for PBI Shredder SG3 01 Shredder Tube Cap Shredder Tube Cap Tool component of PBI 52130 Tool Shredder_HRR Set 01 Shredder Shredder Accessory Box 71x335x240 mm 52201 Accessory Box inner dimensions for storage and shipping 01 of Shredder accessories Shredder Tubes Shredder Tubes consisting of Shredder 52210 a aemaise a EA Tube FT500 PS with Lysis Disk serrated 01 Shredder Ram and Shredder Screw Cap coral colour Box of 100 1 box is included in the PBI Shredder HRR Set Grn Y Cove rd hi arenes Ta eraon GM benee oga G hararese AW Krs BLOGI I beptrens gF Serene ronpeheruiis rna a beinrg one irira mpri shihe mareren prian Eas bashen LIE 398 277 Shredder Shredder Tubes Metal consisting of 52220 Tubes Metal Shredder Tube FT500 PMS with Metal Lysis 01 Disk serrated Shredder Ram and Shredder Screw Cap coral colour Box of 100 1 box is included in the PBI Shredder HRR Set Forceps stainless Forceps stainless Steel st
5. PNetO3 02 Selected media and chemicals for respirometry with mitochondria and permeabilized cells Mitochondr Physiol Network 3 2 MiPNeti1 05 Isolated mitochondria or permeabilized tissues and cells Mitochondr Physiol Network 11 5 MiPNet14 13 Mitochondrial respiration medium MiRO6 Mitochondr Physiol Network 14 13 6 Author contributions and publication versions E ga Prepared by Draxl A Eigentler A and Gnaiger E in February 2012 DA performed the experiments e Version 1 2012 02 29 2012 03 14 e Version 2 2013 01 15 Contribution to K Regio project MitoCom Tyrol funded in part by the Tyrolian Government and the European Regional Development Fund ERDF www oroboros at MitoCom Tyrol OROBOROS INSTRUMENTS OROBOROS Oxygraph 2k http www bioblast at index php PBI Shredder HRR Set PBI Shredder HRR Set Auxiliary HRR Tool for tissue homogenate preparation the Shredder Kit Box contains the heavy duty high torque SG3 driver with convertible handle SG3 base with 3 Description position force setting lever FSL battery charger two lithium ion batteries Shredder Tube Cap Tool The PBl Shredder HRR Set includes the Shredder Kit Box with 100 Shredder Tubes 100 Shredder Tubes Metal a pair of sharp forceps for tissue dissection and a pair of scissors Product ID 13200 02 Link PBl Shredder OROBOROS O2k Catalogue PBl Shredder Purchase Order OROBOROS PBlI Shredder HRR Set consists of H Title
6. al inserts gave comparable results Optimization of homogenization with various tissues will be possible using either type of Shredder Tubes force settings and duration of shredding 2 Materials and Chemicals 2 1 Components of the PBI Shredder HRR Set http www bioblast at index php PBI Shredder_HRR Set e PBI Shredder SG3 stored in the Shredder Kit Box with torque driver and convertible handle metal SG3 base use pre chilled after storage in the fridge with 3 position force setting lever FSL battery charger and two lithium ion batteries Figure 1 e Shredder Tube Cap Tool Figure 2 e Shredder Tube Ram Tool OROBOROS INSTRUMENTS OROBOROS Oxygraph 2k MiPNet17 02 PBI Shredder HRR Set 3 e Box of 100 Shredder Tubes FT500 PS with plastic lysis disk with Shredder Rams and Shredder Screw Caps use pre chilled Figure 3 e Box of 100 Shredder Tubes Metal FT500 PMS with metal lysis disk with Shredder Rams and Shredder Screw Caps use pre chilled Figure 3 e Pair of dissecting forceps stainless steel antimagnetic sharp straight tips e 1 pair of dissecting scissors straight tip sharp front 2 2 Other materials e Microbalance Mettler Toledo 0 01 mg display http www bioblast at index php Microbalance Set Petri dish and 12 well tissue culture plate 50 ml Falcon tubes 1 per Shredder Tube 500 ul pipette with tips Cap ofa 1 5 ml Eppendorf tube cut off Filter paper or soft tissues Timer 1 60
7. and 20 for the second O2k Chamber 23 Insert the stoppers loosely into the O2k Chambers and allow the homogenate to warm up to the experimental temperature for approximately 3 minutes 24 Close the chamber and siphon off the excess of respiration medium 3 5 Detailed protocol Tissue homogenization shredding Immediately after reading the wet weight the samples are transferred to the narrow Ram side of the pre chilled Shredder Tube already capped with the Shredder Screw Cap using the Shredder Tube Cap Tool again Figure 3 and containing 500 ul respiration medium e g MiRO6 or MiRO6Cr using the pair of straight dissection forceps wetted with respiration medium The tissue samples are then cut into smaller pieces with a sharp pair of scissors and evenly distributed on the Lysis Disk at the narrow Ram side of the Shredder Tube The total volume of sample and respiration medium during shredding should not exceed 0 7 to 0 8 ml this prevents medium from being forced into the threads of the cap where it might be lost during uncapping OROBOROS INSTRUMENTS www oroboros at MiPNet17 02 PBI Shredder HRR Set 6 After evenly distributing the small tissue pieces on the Lysis Disk at the narrow Ram side of the Shredder Tube a serrated Shredder Ram is inserted with a twisting motion to press the sample between the serrated surface and the Lysis Disk by using the Shredder Tube Cap Tool Figure Tube Cap Tool 2 Cap As the
8. and turn on the SG3 Driver to seat the Driver bit into the crenellations of the Cap With one hand press down the Driver and with the other hand set the lever into the appropriate position for the sample OROBOROS INSTRUMENTS OROBOROS Oxygraph 2k MiPNet17 02 PBI Shredder HRR Set 5 12 13 14 Activate the Shredder for 10 seconds at position 1 weakest followed by 5 seconds at position 2 stronger this accounts for mouse and trout heart as well as trout liver Remove the homogenate by using the Shredder Tube Cap Tool to unscrew the Shredder Screw Cap from the Shredder Tube by anticlockwise rotation Transfer the sample into a 50 ml Falcon on ice using a 500 ul pipette 15 Rinse the tube with fresh cold respiration medium to recover any residual sample and add to the homogenate 16 Open the Shredder Tube by using the Shredder Tube Ram Tool to open the narrow side of the Shredder Tube 17 Wash any residual tissue out of the tube into the 50 ml Falcon using fresh cold respiration medium 18 Rinse with 4 5 ml respiration medium in total to have 5 ml end volume which is intended for use with two O2k Chambers and keep the sample on ice until used for HRR 19 Siphon off the respiration medium of the O2k Chambers 20 Resuspend the homogenate thoroughly by pipetting 6 times up and down avoiding pipetting on the wall of the tube and any generation of foam 21 Insert 2 5 ml of homogenate into one O2k Chamber 22 Repeat step 19
9. more the oxygen diffusion gradients are reduced compared to permeabilized fibres The PBI Shredder combines a minimum processing time of 10 minutes and easy handling that enables especially beginners to obtain reliable results and the closed Shredder Tubes ensure safety throughout the entire sample preparation process As with any other method training of each individual person with the PBI Shredder improves the handling and the tissue preparation resulting in better results over time as shown with the cytrochrome c test At the beginning of our experiments the cytochrome c effect was larger in cardiac mouse homogenate compared to permeabilized fibres indicating a small degree of functional impairment of myocardial mitochondria is caused by the homogenization process Over the time our skills improved and we were able to diminish the cytochrome c effect in mouse heart homogenate and no cytochrome c effect occurred in mouse liver and mouse brain homogenate The homogenate obtained with this method may contain some tissue particles that are not homogenized but complete cell permeabilization is obtained as evaluated with HRR Therefore a potentially unequal distribution of the homogenate into different O2k chambers can occur due to insufficient resuspension of the homogenate Furthermore a fraction of mitochondria can potentially be lost when insufficient care is taken to retrieve the entire tissue If not OROBOROS INSTRUMENTS www oroboro
10. ne Shredder Tube should be used for one O2k Chamber Place the Eppendorf cap on the microbalance add 100 ul of Biops or respiration medium and tare Transfer the samples with the pair of forceps onto a filter paper During this time of a few seconds wipe off any liquid from the sharp tip of the forceps with another filter paper Then take the samples from the filter paper and touch it Once more shortly onto a dry area of filter paper while holding it with the forceps Afterwards immediately place the samples into the Eppendorf cap and read the wet weight 3 4 Quick protocol 1 10 11 Store the PBI Shredder metal SG3 base and Shredder tubes at 20 C Take a Shredder tube and close the Cap side with a Shredder Screw Cap using the Shredder Tube Cap Tool Add 500 ul of ice cold respiration medium to the Ram side of the Shredder Tube and pre chill the Shredder Tube on ice After reading the Wy transfer the samples to the narrow Ram side of the pre chilled Shredder Tube Cut the tissue samples into smaller pieces with a sharp pair of Scissors Evenly distribute the tissue pieces on the Lysis Disk at the narrow Ram side of the Shredder Tube Close the Shredder Tube with a serrated Shredder Ram Place the filled Shredder Tube into the pre chilled Shredder Base with the Ram side down Twist the Shredder Tube to set the Ram into the holder in the Shredder Base When the tube is seated securely place the SG3 Driver onto the Cap
11. raight Tip sharp 54210 Steel straight for tissue preparation stainless steel 01 Tip sharp antimagnetic One pair is recommended for insertion of the sample into the 02k Chamber and for handling in combination with Forceps stainless Steel rounded Tip sharp Set in HRR Dissection Set and PBI Shredder HRR Set OROBOROS INSTRUMENTS www oroboros at
12. s at MiPNet17 02 PBI Shredder HRR Set 8 all mitochondria are obtained from the tissue it is difficult to evaluate if specific mitochondrial types are enriched or a representative subsample of all mitochondria is obtained If not all mitochondria are obtained from the tissue tissue mass specific mitochondrial respiratory capacity can be measured only on the basis of additional measurements of a mitochondrial marker e g CS activity in the total tissue and in the homogenate to quantify the mt yield and refer respiration of the homogenate to Ww of tissue The application of an additional tool to remove the serrated Shredder Ram as well as the Shredder Screw Cap after homogenization increased the mitochondrial yield by washing out the homogenate completely from both sides of the Lysis Disk 5 References Doerrier VC Draxl A Eigentler A Gnaiger E 2013 Mitochondrial respiration in permeabilized fibres versus homogenate from trout heart and liver Mitochondr Physiol Network 17 03 Gross VS Greenberg HK Baranov SV Carlson GM Stavrovskaya IG Lazarev AV Kristal BS 2011 Isolation of functional mitochondria from rat kidney and skeletal muscle without manual homogenization Analyt Biochem 418 213 223 Pressure BioSciences Inc The Shredder SG3 and Shredder PULSE Tubes Product Specification Sheet 1 2 Pressure BioSciences Inc The Shredder SG3 User Manual 1 16 Mitochonadr Physiol Network MiPNet Manuals and Protocols Mi
13. tissue is shredded the ee Place the filled KA pe Solid tissue sample is placed S h red d e r Tu be n to a pigura S FTS00 PS the pre chilled Ali 3 The vebsting Shredder Pulse Tube for Shredder Base Ram C serrated Ram use with the PBI Shredder side down and twist grinds the tissue d df G t oo om Grosset to set the Ram into the holder in the Shredder Base When the tube is seated securely place the SG3 Driver onto the Cap and briefly turn the driver on in order to seat the Driver bit into the crenellations of the Cap Lysis Disk While pressing down the Driver with one hand set the lever into the appropriate position for the sample For mouse and trout heart as well as trout liver activation of the Shredder for 10 seconds at position 1 weakest followed by 5 seconds at position 2 stronger was evaluated as optimum regime with a maximum of the sample passing through the Lysis Disk into the upper chamber of the Shredder Tube containing functionally intact mitochondria Position 3 strongest was not required in these samples This short processing time does not significantly heat the sample It is recommended to use a timer for application of the shredder 3 6 Removing the homogenate To remove the processed homogenate use the Shredder Tube Cap Tool to unscrew the Shredder Screw Cap from the Shredder Tube by anticlockwise rotation Transfer the Sample into a 50 ml Falcon on ice using a 500 ul pipette To reco
14. ver any residual sample rinse the tube with fresh cold respiration medium and add to the homogenate Use the Shredder Tube Ram Tool to open the narrow side of the Shredder Tube and wash the sample out of the tube with respiration medium Rinse with 4 5 ml in total and at the end there should be 5 ml of homogenate in the Falcon tube on ice This volume is intended for use with two O2k Chambers Keep the sample on ice until used for HRR OROBOROS INSTRUMENTS OROBOROS Oxygraph 2k MiPNet17 02 PBI Shredder HRR Set 7 3 7 Experimental setup with the Oxygraph 2k For experiments with homogenate preparations the medium of the O2k chamber was siphoned off The homogenate was resuspended thoroughly by pipetting 6 times up and down avoiding any generation of foam and 2 5 ml were inserted into one O2k chamber This was repeated for the second chamber The homogenate was then allowed to warm up to the experimental temperature for 3 min without closing the chamber but with the stopper inserted loosely 4 Conclusions The PBI Shredder provides a standardized tissue preparation for obtaining disrupted cells with functional mitochondria that may be used directly for HRR or the homogenization process may be followed by further isolation of mitochondria In addition the homogenate is suitable for optical measurements e g O2k Fluorometry with safranin for detection of mt membrane_ potential where a homogenous suspension is required Further
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