Instrumental background correction, accuracy of oxygen
Contents
1. cases of high experimental oxygen fluxes when background correction is merely within 1 5 of flux Taken together the concept of instrumental background oxygen flux and appropriate corrections are indispensible components of HRR To obtain accurate parameters for instrumental background correction background tests are performed in which several oxygen levels are set in the closed O2k Camber related to the experimental oxygen regime and background oxygen flux is measured as a function of oxygen concentration 2 Preparations 2 1 Solutions Dithionite solution 10 mM in phosphate buffer Addition to 10 ml final 174 1 0 017 g Phosphate buffer 50 mM pH 8 Finalconc Component_ Fw _ _ Addition to 1 liter final NaHPO 2 HO 178 0 7 83 g NaH2PO H2O 138 0 0 81g Dithionite solution has to be prepared fresh and stored on ice immediately before use Add 17 mg dry dithionite into a glass volumetric flask and add phosphate buffer to the final 10 ml volume Keep the flask closed and minimize exposure to air OROBOROS INSTRUMENTS O2k Protocols MiPNet14 06 Instrumental O background 3 2 2 Media The dithionite background experiment has to be performed in MiRO6 or MiRO5 MiPNeti4 13 In many other media including cell culture media and unbuffered water side reactions lead to additional oxygen fluxes which interfere with the instrumental background oxygen flux As an alternative a strongly buffered alkali2. determined initially e Set the Volue Vinject to 5 ul e Test start before inserting the needles to replace the dithionite solution in the needles e Wait for stabilisation of oxygen flux e Inject 5 ul and calculate SF using Eq 1 Example Oxygen level in the chamber is 160 uM The user wants to obtain four background levels in addition to the one recorded near air saturation With four evenly OROBOROS INSTRUMENTS O2k Protocols MiPNet14 06 Instrumental O background 13 Spaced steps it is possible to reach a minimum of 20 UM reducing the oxygen concentration by 35 uM steps The necessary injection volume Vinject to achieve the desired reduction of oxygen concentration can then be calculated from Eq 2 In the present example SF 0 7 ACo2 35 UM Vehamber 2 MI Cnazs204 9 8 mM Vinject 10 Ul Four injections of 10 ul each should therefore bring the oxygen concentration near the desired last level of 20 UM Optionally with a fifth injection zero oxygen concentration could be reached It is recommended to use a larger excess volume for zero calibration Always consider the expected experimental oxygen concentration range For an experiment at high oxygen levels calculate injection to decrease from the initial oxygen level e g 350 uM to the final oxygen concentration e g air saturation The minimum time required between injections to obtain stable fluxes is about 10 minutes The time course of the instrumen
3. main menu select TIP select BG Feedback W from the dropdown menu and press Load setup Start the titration programme During operation the TIP2k window may be closed The TIP2k programme starts allowing for a delay of 1200 s 20 min during which time oxygen flux can Stabilize after closing the chamber providing the first background level J 1 Then the first injection starts at 0 5 ul s The TIP2k operates now in feedback mode while oxygen levels decline The TIP2k stops when an O2 concentration lt 100 uM is reached and possibly overshoots by 10 uM to yield a level of about 90 uM J 2 The 1200 s interval 20 min is programmed as a feedback control time of 300 s plus a delay of 900 s before each further injection at 0 25 ul s to 50 uM J 3 and 0 1 ul s to 23 uM J 4 reducing the overshoot to 5 uM and 3 uM After recording the last background level J 4 at 20 uM a final titration of excess dithionite is induced in the direct control mode for a zero oxygen calibration of the OroboPOS OROBOROS INSTRUMENTS O2k Protocols MiPNet14 06 Instrumental O background 7 3 2 Manual injections SF Ano2 eff Ano2 calc ACo Vi chamber Vinject Cna2S204 Use a Hamilton syringe for manually injecting the dithionite solution The effective concentration of dithionite decreases in the stock solution over time due to autoxidation when small amounts of oxygen leak into the solution The potency of the solution
4. OROBOROS INSTRUMENTS high resolution respirometry Mitochondrial Physiology Network 14 06 03 1 8 2014 2009 2014 OROBOROS O2k SOP B www bioblast at index php MiPNet14 06 InstrumentalBackground Instrumental background correction accuracy of oxygen flux and SOP Fasching M Gnaiger E OROBOROS INSTRUMENTS Corp high resolution respirometry Schopfstr 18 6020 Innsbruck Austria Email erich gnaiger oroboros at www oroboros at Section We TMOU oN eaa a tie ciaaig dees iv aden aca ieanaaie bas ede N 1 2 Preparations sssssssensnesnsnesunossunonnnennnasunsesunossnsennnennnasnnnesnnennnne 2 ZA ONON eaaa E E EE E maesdananecotaameeessaaans 2 22 MOO aE E E E EE E E E EE EEEE 3 2 3 Calibration of the oxygen SENSOSS ssssessssssssrsrrsrrrrrrrnerrrerenere 3 2 4 Experimental oxygen concentration s sesesesssssrrrrsrrrrrrnrnrnrnrnrne 3 3 Instrumental background teSt ssssesssssrssrsrrsrrnrrrrsnerrrrrnrrrrerenere 5 3 1 TIP2k in feedback control MOdE cece 5 3 2 Manual INJeCUCiSisscascecsascusssescovseneananesoscepananaeacssmnreansaaweeqnaaneans 7 4 Analysis and calculation of background parameters s s ssssessssssreresas 8 Sa RelerenCES cee wana saasmeecs scans nore a E NE EEA RASET A 8 Supplement A Background parameters and accuracy of flUX 006 9 Oxygen consumption by the polarographic oxygen SENSOLP ceseeeeeees 9 Accuracy of instrumental background tests cccccceee
5. alculated for the intercept a and the slope b by linear regression for each individual chamber The full and stippled lines show the linear regression and 99 confidence intervals calculated through all data points Flux J pmol s ml Roz is about 9 V at air saturation 37 C and a gain of 4 10 V A and is thus typically 2 2 uA under these conditions In the cathode reaction Eq 3a electric flow Ie A C s is stoichiometrically related to molar oxygen flow Io2 mol O2 s through the stoichiometric charge number of the reaction ve yo2 4 and the Faraday constant F i e the product of the elementary charge and the Avogadro constant F 96 485 53 C mol Mills et al 1993 The oxygen electric flow ratio is Gnaiger 1983 OROBOROS INSTRUMENTS O2k Protocols MiPNet14 06 Instrumental O background 10 Yoze Ve o2 F 4 96 485 mol C 5 2 591068 10 mol O2 C 2 591 pmol O2 s yA Oxygen consumption by the POS can be directly measured in the closed Oxygraph chamber at air saturation Fig 2 as volume specific oxygen flux Jo2 pmol s cm and the corresponding theoretical oxygen flux in Eq 3a can be calculated Jo2 pos Fig 3 Jo2 pos Roz Roz 0 lt Yore Forze V 6a where Roz is the raw signal at zero oxygen zero current and V is the chamber volume of the Oxygraph 2k 2 cm Figure 3 Instrumental background oxygen flux J o2 as a functi
6. can be tested by injecting a small volume 5 ul into the closed oxygraph chamber and observing the change in oxygen concentration The stoichiometric correction factor SF expresses the deviation of the effective dithionite concentration from the dithionite concentration added initially _ Ano eff _ ACos Venamber 1 SF Ang calc Vinjet C Na S 0 Stoichiometric correction factor for dithionite concentration Effective change of the amount of oxygen umol Calculated change of the amount of oxygen umol Effective drop in oxygen concentration umol dm umol Chamber volume cm ml Injected volume of dithionite solution mm ul Dithionite concentration in the initial stock solution approx 9 8 mmol dm considering a complete consumption of oxygen originally dissolved in the aqueous solvent irrespective of further oxygen uptake by the effectively anoxic solution Vinject IS the volume injected to achieve a specific drop in oxygen concentration AC oz V ta inject 2 SF CNa S 0 A typical value of SF is 0 7 in a freshly prepared stock solution Since no accurate oxygen concentrations have to be achieved for determination of an instrumental background a value of 0 7 can be used for most purposes When using the TIP2k in Feedback Control Mode calculation of SF is not necessary OROBOROS INSTRUMENTS O2k Protocols MiPNet14 06 Instrumental O background 8 4 Analysis of instrumental backg
7. e Clark type polarographic oxygen sensor POS yields an electrical Signal while consuming the oxygen which diffuses across the oxygen permeable membrane to the cathode The cathode and anode reactions are respectively O 2H20 4e 74 40H 3a 4 Ag gt 4Ag 4e 3b 4 Ag 4Cl 4 AgCI 3b The electric flow current Je A is converted into a voltage electric potential Va V and amplified In the Oxygraph 2k the gain Fo26 can be selected in DatLab within the Oxygraph setup menu with values of 1 2 4 or 8 10 V A where 1 V A is the basal gain at a gain setting of 1 The raw signal after amplification Roz V is related to the original POS current 1 Ia Roz Fo2 G 4 Figure 2 Instrumental 3 a 2 06 0 39 4 o be 0 0256 0 0028 3 background oxygen flux J o2 r 0 93 Py as a function of oxygen concentration Co2 uM in the OROBOROS Oxygraph 2k 37 C NaCl solution with an oxygen solubility factor of 0 92 relative to pure water Measurements in 52 chambers 2 ml volume of 26 different instruments In all tests four oxygen ranges were selected consecutively in 0 30 100 150 200 declining order Each oxygen Oxygen concentration uM concentration was maintained for 20 min at the end of which time intervals of 200 seconds corresponidng to 200 data points at the sampling interval of 1 s were chosen for estimating average flux at each corresponding oxygen concentration Averages and SD were c
8. eeeeeeeeeeees 11 Supplement B TIP2k in direct control MOE cceeeee cece eeeeeeeeeeeenees 12 1 Introduction For calibration of the polarographic oxygen sensor POS and measurement of instrumental background oxygen consumption incubation medium without biological sample is added into the O2k Chamber at experimental conditions In a closed chamber under these conditions ideally oxygen concentration remains constant In practice however instrumental background effects are caused by backdiffusion into the instruments oroboros at www oroboros at MiPNet14 06 Instrumental O background 2 chamber at low oxygen pressure oxygen diffusion out of the chamber at elevated oxygen levels and oxygen consumption by the polarographic oxygen sensor OroboPOS Determination of instrumental background constitutes an important standard operating procedure SOP in high resolution respirometry HRR Instrumental background oxygen flux is i minimized in the OROBOROS Oxygraph 2k by instrumental design and selection of appropriate materials In addition ii instrumenal background is routinely tested and iii background correction of oxygen flux is applied automatically by DatLab As an important component of quality assurance instrumental background is monitored at regular intervals during an experimental project and documented as a standard operational procedure to exclude instrumental artefacts This SOP _ is implemented even in
9. g the equilibration process After Stabilisation of oxygen flux the first state of background flux is recorded by marking an appropriate section of the oxygen flux MiPNet19 01E Further steps of oxygen levels towards air saturation may be achieved by shortly opening the stopper again using the stopper spacer tool 2 observing the drop of oxygen concentration and closing the chamber at the desired oxygen level Preferentially use the TIP2k method described below OROBOROS INSTRUMENTS O2k Protocols MiPNet14 06 Instrumental O background 5 3 Instrumental background test 3 1 TIP2k in feedback control mode Fill the TIP2k syringes with the freshly prepared dithionite solution rinsing the syringes at least once with the dithionite solution and taking care to minimize exposure of the dithionite solution to air Use a large volume glass syringe and long needle to fill both TIP2k syringes sequentially After air calibration close the chamber either directly normoxia or after elevating oxygen levels hyperoxia After closing the chamber insert the TIP2k needles through the stopper TIP2k Manual go Bioblast MiPNet12 10 TIP2k Manual 5 a 8 9 20 E 44 150 gt a 0 100 k 4 x D Z e Q Z 8 S v 0 2 0 00 20 1 02 30 23 44 2 05 00 2 g Ranne h min s a 05 ae clitip in TIP Stop TIP Stop i Stop P004 J a J Qed J A Oo TIP2k Setup BG _ Feedback I
10. ne phosphate buffer may be used gt 100 mM gt pH 8 Results obtained in MiRO6 can be used for other media e g cell culture media 2 3 Calibration of the oxygen sensors Be go Bioblast MiPNet06 03 POS Calibration SOP 2 4 Experimental oxygen concentration Graded levels of oxygen can be achieved in instrumental background tests with the aid of a gas phase included in the O2k Camber replacing air with nitrogen or argon to decrease oxygen levels or with oxygen to increase oxygen levels Mass transfer between gas and liquid phases proceeds until the targeted oxygen level is reached This process is stopped when the gas phase is eliminated by closing the chamber Gnaiger et al 1995 Gnaiger 2008 The main disadvantage of intermittently opening the O2k Chamber for application of a gas phase during background experiments is the risk of inclusion of gas bubbles when closing the chamber Elimination of gas bubbles is more difficult in O2k MultiSensor ISE applications when one or two additional electrodes are introduced through inlets in the stopper Importantly in these applications instrumental background correction is even more important since inserted electrodes add new oxygen storage capacities and potential leaks An automatic O2 background test has been introduced to overcome these problems with the TIP2k Instrumental background tests should cover the entire experimental oxygen range Most experiments are performed a
11. nstrumental background oxygen flux at air saturation 176 uM 37 C 600 m altitude 90 uM 45 uM 20 uM Each level was maintained for 20 minutes The following parameters are used in the set up file Line Mode Start injection if Stop injection if Flow Delay Interval Volume oxygen level left oxygen level left or chamber is gt right chamber is lt iis sr are pa o os pao S ere fo a jw jo s qe jo J ow o S awp S 0 OROBOROS INSTRUMENTS O2k Protocols MiPNet14 06 Instrumental O background 6 Control Chemicals Configuration Info Delay s 1200 00 20 00 Feedback control Stop and next program line after J0 seconds or 8 cycles 0 for unlimited Mode Direct control Feedback control Select f TIP backward 7 VoF Youre MA uay o VolFlom Vome lu A e Tower Pause ti woltTime Flow p s 5 508 7 O2 Concentration Pw mol gt 2 Start 02 Concentration PA r 1 Sto FlowsTime Time 200 09 15 ee E Feedback line Delete Insert Prograrr line Inzert Replace l Delete LU Move up Move down aeee Vol ul Flow ul EOE bs o e s 0 500 ZULU gt F 300 60 000 0250 200 00 O ae vw anal gt 60 00 2s shart 900 20 000 0 100 200 00 02 Concentration Pw nmol l gt 30 00 2 start 900 100 000 50 000 2 00 Feedback control cannot predict final volume BG_Feedback Load setup Save setup Hide details Cancel Close In the DatLab
12. on of the theoretical oxygen consumption by the polarogrpahic oxygen sensor POS calculated from the electrical signal current as a function of oxygen concentration from data in Figure 1 The line of identity dashed illustrates the full correspondence between experimental and theoretical 0 1 2 3 oxygen consumption at air Expected POS flux J pos pmol st mit saturation top right and the increasing deviation at declining oxygen concentration owing to a linear increase of oxygen backdiffusion Flux J pmol s ml It is more convenient to relate the theoretical oxygen consumption of the POS to the measured oxygen concentration Coz UM using the oxygen calibration factor Fo2 UM V Jo2 pos Coz Foz Youe Foza V 6b Combining constants from Eq 5 at a gain setting of 4 V A and a volume of 2 cm Eq 6 is Jo2 pos Ro2 Ro2 0 0 3239 pmol s cm7 V 6c Coz Fo2 c 0 3239 pmol s cm V Figure 4 Noise SD of the mean of the apparent oxygen flux J o2 as a function of noise SD of the mean of oxygen concentration Coz 180 2 uM at 95 1 kPa barometric pressure in the open chamber of the OROBOROS Oxygraph 2k 37 C NaCl solution at air saturation over time intervals of 200 seconds corresponidng to 200 data points at the sampling interval of 1 s Each data point V 43 represents an independent Oxygraph 2k chamber 2 ml volume The SD of oxygen concentration was calc
13. pplied Oxygen concentration at air saturation Co2 179 9 UM Average oxygen concentration at J 1 Coz1 177 2 UM Oxygen calibration signal at air saturation Roz 8 744 V Gain 4 Oxygen calibration signal at zero oxygen Rozo 0 033 V Gain 4 Oxygen calibration factor Foz 20 69 UM V Jo2 pos 0 3239 x 177 2 20 69 2 77 pmol s cm gt At air saturation in the 2 cm chamber the theoretically expected oxygen consumption by the sensor is 2 77 pmol s cm in direct agreement with the experimental result At an average flux of 2 64 pmol s cm 0 35 SD N 52 Fig A2 the ratio between measured and theoretically expected oxygen consumption by the POS was 0 95 0 12 SD N 52 This provides possibly the first experimental evidence for the exact 4 electron stoichiometry in the reduction of oxygen at the cathode of the POS Supplement B TIP2k in direct control mode wes TIP2k Manual LA go Bioblast MiPNeti2 10 TIP2k Manual Fill the TIP2k syringes with freshly prepared dithionite solution After air calibration record the first point of the background experiment as described above Programming the TIP2k Calculate the necessary injection volumes as described in Section 2 5 initially assuming SF 0 7 stoichiometric correction factor for dithionite concentration SF can be calculated after the first injection and if necessary the TIP2k be reprogrammed for subsequent injections Alternatively SF may be
14. round tests go Bioblast Zi jx MiPNet19 01E MiPNet08 09 MiPNet10 04 Gnaiger 2001 22 2008 22 Gnaiger et al 1995 2 5 References Gnaiger E 2008 Polarographic oxygen sensors the oxygraph and high resolution respirometry to assess mitochondrial function In Mitochondrial Dysfunction in Drug Induced Toxicity Dykens JA Will Y eds John Wiley 327 352 gt 2 Gnaiger E 2001 Bioenergetics at low oxygen dependence of respiration and phosphorylation on oxygen and adenosine diphosphate supply Respir Physiol 128 277 297 22 Gnaiger E Steinlechner Maran R M ndez G Eberl T Margreiter R 1995 Control of mitochondrial and cellular respiration by oxygen J Bioenerg Biomembr 27 583 596 22 f A O2k Manual MiPNet19 01E O flux analysis real time gt MiPNet12 10 Titration Injection microPump TIP2k user manual 02k Protocols MiPNet06 03 POS calibration accuracy and quality control SOP MiPNetO8 09 HRR with leukemia cells respiratory control and coupling gt MiPNet10 04 HRR Phosphorylation control in cell respiration gt MiPNet14 13 Mitochondrial respiration medium MiRO6 a A Full version go Bioblast MiPNet14 06 InstrumentalBackground OROBOROS INSTRUMENTS O2k Protocols MiPNet14 06 Instrumental O background 9 Supplement A Background parameters and accuracy of flux Oxygen consumption by the polarographic oxygen sensor Th
15. t oxygen levels below air saturation but high oxygen levels are used with permeabilized fibres H202 With MiRO6 containing catalase oxygen concentration is easily adjusted by injecting small amounts of a H202 stock solution into the closed chamber MiPNet14 13 OROBOROS INSTRUMENTS O2k Protocols MiPNet14 06 Instrumental O background 4 Oxygen levels are increased in steps of lt 200 uM e g from air saturation up to 350 uM to prevent formation of gas bubbles in the medium O gas phase For oxygen concentrations above 400 uM the preferred approach is application of a gas phase with high oxygen pressure If a calibration at air saturation was just performed there is already an open chamber i e a chamber with a gas phase Insert the stopper completely closing the chamber Siphon off any medium extruded through the stopper capillary Then partially open the stopper arrow 1 insert the stopper spacer tool 2 and push down the stopper 3 The gas injection syringe with supplied needle 4 correct length and spacer 5 is filled with oxygen gas Inject a few ml of oxygen into the gas phase 6 thereby creating an elevated oxygen pressure above the stirred aqueous medium Oxygen in the gas and aqueous phases will start rapidly to equilibrate Observe the oxygen signal in DatLab carefully When the desired oxygen concentration is nearly reached close the chamber thereby extruding the gas phase and stoppin
16. tal background should match the decline of oxygen concentration in the real experiment Longer intervals will typically be chosen 15 min in our example The TIP2k can be set up in the following way Select Direct control and Vol Flow Delay s 0 Volume ul E Flow ul sec Interval s 900 Cycles 4 Start the experiment with Start OROBOROS INSTRUMENTS O2k Protocols
17. terval of 1 s Each data YM 0 00 point N 43 represents an 0 50 100 150 200 independent Oxygraph 2k Oxygen concentration uM chamber 2 ml volume Flux was calculated from concentration smoothed with a moving average 30 data points using an eight point polynomial for calculation of the slope The full and stippled lines show the linear regression and 99 confidence intervals To express noise of flux independent of small changes in flux over time linear regressions were calculated through 200 second sections and this trend was subtracted from flux before calculating the SD Accuracy of instrumental background tests Instrumental background interferes with accurate measurement of respiratory oxygen flux if background effects remain undefined The instrumental oxygen background parameters are a property of the O2k Chamber Any contamination of the medium causing oxidative processes microbial respiration is detected Then background oxygen consumption is a property of a contaminated medium Otherwise instrumental background does not depend on the specific medium Therefore OROBOROS INSTRUMENTS O2k Protocols MiPNet14 06 Instrumental O background 12 background parameters obtained in one medium can be used for another medium in the same chamber In a series of 52 experimental background determinations 52 different O2k chambers 2 ml volume 37 C were tested Oxygraph 2k Series A The following average conditions a
18. ulated from the raw signal without smoothing Flux was calculated from concentration smoothed with a moving average 30 data points using an eight point polynomial for calculation of the Slope The outlier full circle corresponds to a data set with an individual spike OROBOROS INSTRUMENTS O2k Protocols MiPNet14 06 Instrumental O background 11 The full and stippled lines a te show the linear regression 10 Air saturation open and 99 confidence N intervals On average signal 08 stability was indicated by 2 apparent oxygen fluxes close to zero during air calibration when oxygen concentration is maintained stable by exchange with the gas phase Average J o2 amounted to 0 04 0 14 pmol s cm range from 0 28 to 0 25 0 00 0 01 0 02 0 03 0 04 0 05 0 06 0 07 pmol s cm To express Signal noise independent of SD of oxygen concentration uM these low levels of signal drift linear regressions were calculated through these 200 second sections and this drift was subtracted from the concentration before calculating the SD O2 SD of flux J O NO 1 00 Figure 5 Noise SD of the EE E mean of the instrumental T Slope 0 0031 background oxygen flux 2 0 75 J o0 aS a function of oxygen concentration Co2 Q uM in the OROBOROS 56 0 50 Oxygraph 2k 37 C NaCl gt solution over time S intervals of 200 seconds 0 25 corresponidng to 200 data points at the sampling Q in
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