Oroboros Open Support alert

Oroboros Open Support alert - 2024

Dec 2024

Dec-30 Mo

Why use a Cover-Slip?

Placing a Cover-Slip on the Stopper minimizes contamination and evaporation. During optical measurements in the Oroboros, using the black Cover-Slip prevents light penetration and disturbance of the fluorescence signal.
- »Cover-Slip/black«
- communicated by »Leo Elettra« and »Cecatto Cristiane«

Dec-26 Th

"When using a syringe with a chemical different from the one usually assigned to the syringe, repeated washing steps and leaving the syringe filled with absolute ethanol overnight may be crucial to prevent contamination by carry-over."

- »MiPNet19.14 SOP Hamilton microsyringes«
- communicated by »Baglivo Eleonora« and »Cecatto Cristiane«

Dec-12 Th

"When working with potentially sticky tissue, stop the stirrer, siphon off H₂O, remove the stirrer (...) clean the stirrer bar with a paper towel and rinse it with H₂O. Return to the same chamber."

- »MiPNet19.03 O2k-cleaning and ISS«
- communicated by »Cardoso Luiza« and »Cecatto Cristiane«

Nov 2024

Nov-18 Mo

Can Amplex® UltraRed be used to measure ROS production in all tissue types and preparations?

Not all tissue types and preparations can be used. Liver homogenate cannot be used with the Amplex® UltraRed assay due to the cytosolic components of liver tissue which interfere with the measurement. Isolating liver mitochondria overcomes this drawback.
- »Amplex UltraRed« »Tissue homogenate«
- communicated by »Willis Jaime R« and »Leo Elettra«

Nov-14 Th

"The instrumental background tests must be carried out with the Q-Stopper and reference electrode in place. The values obtained with the standard stoppers for respirometry cannot be used for Q-Sensor experiments."

- »MiPNet24.12 NextGen-O2k: Q-Module«
- communicated by »Grings Mateus« and »Schmitt Sabine«

Nov-04 Mo

Glass chamber stuck in the Oroboros?

Spilled medium may cause adhesion between the Oroboros chamber and copper block. Wash with warm water while the Oroboros is on and heated to 37 °C. Then, gently screw in the sensor holder to push the chamber upward.
- »chamber#Oroboros chamber_disassembly Oroboros chamber disassembly«
- communicated by »Timon-Gomez A« and »Baglivo E«

Oct 2024

Oct-31 Th

Get a more in-depth look into mitochondrial function with fluorometry. The Oroboros Fluo Smart-Module enables the assessment of ROS production (H₂O₂ flux; Amplex UltraRed), membrane potential (Safranin, TMRM, Rhodamine 123), calcium uptake (CaG), and ATP production (MgG)

- »MiPNet28.09 Oroboros Fluo Smart-Module Manual« »Fluorometry«
- communicated by »Willis Jaime R« and »Schmitt Sabine«

Oct-22 Tu

How is data from the mark statistics window in DatLab 8 exported in the correct format for analysis using the O2 analysis template?

In the mark statistics window (F2), check the option "Export for DL analysis template" and select "O2 analysis template" in the drop-down menu. Copy to the clipboard and paste your data in to the corresponding Excel sheet.
- »Mark statistics - DatLab«
- communicated by »Grings M« and »Willis Jaime R«

Oct-17 Th

"Any moisture on the mesh increases the risk of air bubbles being trapped below the Sample Holder after closing the Oroboros chamber."

- »MiPNet27.07 Sample Holder«
- communicated by »Leo Elettra« and »Timon-Gomez Alba«

Oct-07 Mo

Do you know all keyboard shortcuts for the DatLab software?

DatLab provides several shortcuts for quick access to functions and settings without the need for a mouse.
- »Keyboard shortcuts - DatLab«
- communicated by »Baglivo Eleonora« and »Willis Jaime R«

Oct-03 Th

"It is important to have the correct Oroboros chamber volume entered for calculations of oxygen flux. Entries can be edited at any time during real-time or post-experiment analysis."

- »MiPNet24.14 Oroboros sV-Module Manual«
- communicated by »Timon-Gomez Alba« and »Willis Jaime R«

Sep 2024

Sep-24 Tu

The oxygen concentration is unusually high or low when starting a new measurement?

When starting a new measurement, the oxygen concentration is calculated by DatLab based on the default values or the most recently applied air calibration. If the temperature, barometric pressure, or solubility factor differ between the actual measurement and the default settings or the most recently performed and applied air calibration, the calculated oxygen concentration can be very different from the actual oxygen concentration.
- »Oxygen signal«
- communicated by »Schmitt Sabine« and »Willis Jaime R«

Sep-19 Th

"When working at low oxygen levels it is recommended to measure R0 before and after each measurement using the experimental sample for zero calibration."

- »MiPNet06.03 POS-calibration-SOP«
- communicated by »Baglivo Eleonora« and »Leo Elettra«

Sep-05 Th

"In the presence of high catalase activity, hydrogen peroxide, H₂O₂, is titrated into the Oroboros chamber to increase oxygen levels"

- »OroM2.1 MiR05-Kit«
- communicated by »Cardoso Luiza« and »Baglivo Eleonora«

Aug 2024

Aug-22 Th

"Once a polarization voltage higher than 0 mV is selected and applied [in ‘Start measurement’ or ‘Oroboros Control (active)’ window], the LED of the Fluo sensor lights up."

- »MiPNet28.09 Oroboros Fluo Smart-Module Manual«
- communicated by »Grings Mateus« and »Cardoso Luiza«

Aug-19 Mo

Do I need to measure muscle tissue respirometry immediately after the biopsy?

No, the sample can be stored in ice-cold BIOPS solution at 0 °C for several hours, depending on the source of muscle tissue.
- »MiPNet14.14 PermeabilizedFiberPreparation«
- communicated by »Willis Jaime R« and »Timon-Gomez Alba«

Aug-08 Th

"Instrumental background tests should cover the entire experimental oxygen range."

- »MiPNet14.06 Instrumental O2 Background«
- communicated by »Schmitt Sabine« and »Baglivo Eleonora«

Aug-05 Mo

How do I ship my Oroboros?

To avoid any damage, the Oroboros Main Unit must be shipped without Oroboros chambers and POS in the Packing\Oroboros Box 1, as only this packing material provides sufficient protection during transport. Be sure to follow the general packing instructions.
- »Shipping an Oroboros«
- communicated by »Timon-Gomez Alba« and »Grings Mateus«

Jul 2024

Jul-25 Th

"Oxia generates gaseous oxygen and hydrogen (...) used to control the O₂ regime in the Oroboros chamber"

- »MiPNet26.14_Oxia«
- communicated by »Cardoso Luiza« and »Schmitt Sabine«

Jul-22 Mo

Do you know how to export data points from your DatLab files?

Export all information and events from selected plots to a text file (*.csv) for further use in Excel and other programs compatible with .csv extension.
- »Export as CSV - DatLab«
- communicated by »Baglivo Eleonora« and »Grings Mateus«

Jul-11 Th

Cyclic voltammetry (CV) is a type of electrochemical measurement, applied as a quality control step to (1) determine the redox potentials of coenzyme Q2, (2) check the quality of the Q-sensor, and (3) test the interference of chemicals used in the HRR assay with the Q-sensor.

- »MiPNet24.16 DatLab8.0: CV-Module«
- communicated by »Leo Elettra« and »Baglivo Eleonora«

Jul-08 Mo

How do you avoid clogged Hamilton syringes?

Perform cleaning cycles immediately after use to avoid precipitate formation, especially with syringes used for ADP (with MgCl₂) and fatty acids. In case of clogged syringes, follow the recommendations in the MiPNet below.
- »MiPNet19.14 SOP Hamilton microsyringes«
- communicated by »Grings Mateus« and »Timon-Gomez Alba«

Jun 2024

Jun-27 Th

NAD-redox ratios are expressed as the reduced NADH-fraction. The reduced NADH-fraction is calibrated for minimum and maximum NADH fluorescence values, which correspond to the fully oxidized and fully reduced states of the NAD pool, respectively.

- »MiPNet26.12_NextGen-O2k:_NADH-Module«
- communicated by »Leo Elettra« and »Grings Mateus«

Jun-24 Mo

Maintain a record of measurement information using the experimental log of your DatLab file.

The experimental log is generated for each measurement and provides detailed information about the experimental run that can be saved as PDF or printed for quality control.
- »Experimental log - DatLab«
- communicated by »Baglivo Eleonora« and »Grings Mateus«

Jun-13 Th

The glassy carbon and platinum electrode of the Q-sensor are tightly built-in into the Q-Stopper and need to be polished and cleaned before each experiment.

- »MiPNet24.12_NextGen-O2k:_Q-Module«
- communicated by »Schmitt Sabine« and »Cardoso Luiza«

Jun-10 Mo

The oxygen signal is a flatline?

If there is no response of the oxygen signal after the stirrer test or titration of dithionite, the raw signal might be at the detection limit. Check the raw signal, which should be between 1 and 3 µA at air saturation, at 25-37 °C and with a gain of 1.
- »Oxygen_signal«
- communicated by »Schmitt Sabine« and »Willis Jaime R«

May 2024

May-30 Th

"Harmonization of isolation protocols yielding intact and enzymatically active mitochondria are critical to assess mitochondrial respiratory function"

- »MiPNet20.06 Isolation mouse heart mitochondria«
- communicated by »Cardoso Luiza« and »Leo Elettra«

May-27 Mo

Same experimental conditions, but different O₂ fluxes in the left and right chambers?

Check for instrumental and operational factors that could be causing O₂ flux differences between the Oroboros chambers.
- »Different O2 fluxes in left and right chamber«
- communicated by »Timon-Gomez Alba« and »Baglivo Eleonora«

May-16 Th

"The sample holder protects (...) fragile samples from potential damage caused by stirring the medium."

- »MiPNet27.07 Sample Holder«
- communicated by »Willis Jaime R« and »Timon-Gomez Alba«

May-13 Mo

Do you see fluctuations of O₂ slope negative following a pattern similar to the Peltier power, but without block temperature variation?

Placing the Oroboros under direct airflow of an air conditioning equipment may lead to disturbances in the O₂ slope negative.
- »Fluctuations of O₂ slope negative«
- communicated by »Cardoso Luiza« and »Grings Mateus«

May-02 Th

"In respirometric studies on blood cells, the purity of cell preparation should always be emphasized and documented."

- »MiPNet21.17 BloodCellsIsolation«
- communicated by »Timon-Gomez Alba« and »Baglivo Eleonora«

Apr 2024

Apr-29 Mo

How can I update DatLab 8 to the latest version?

If your computer with DatLab 8 is connected to the internet, go to “Check for updates” in the Help menu of DatLab 8, and follow the simple installation instructions.
- »Updating - DatLab«, »DatLab 8«
- communicated by »Leo Elettra« and »Baglivo Eleonora«

Apr-18 Th

How to store the POS overnight and up to several months?

Fill the chamber and the receptacle of the loosely inserted stopper with 70 % EtOH, add the Cover-slip and switch off the Oroboros.
- »MiPNet19.03 O2k-cleaning and ISS«
- communicated by »Leo Elettra« and »Grings Mateus«

Apr-15 Mo

Do you observe either a zero or low raw O₂ signal when starting a measurement?

Perform a test to evaluate whether this is related to an improperly positioned chamber or a faulty connection between the OroboPOS and the OroboPOS-Connector.
- »Oxygen signal«
- communicated by »Grings Mateus« and »Schmitt Sabine«

Apr-04 Th

Why is it important to check the H₂O₂ sensitivity when conducting an Amplex UltraRed (AmR) assay?

The sensitivity is influenced by experimental time, medium composition, added chemicals and sample, requiring multiple H₂O₂ calibrations throughout the measurement. After the initial AmR calibration, ensure that the sensitivity is within the recommended range.

- »MiPNet24.10 H2O2 flux analysis«, »Amplex UltraRed«
- communicated by »Grings Mateus« and »Schmitt Sabine«

Mar 2024

Mar-21 Th

How well do you handle your Hamilton syringes? What is a cleaning cycle?

"A syringe cleaning cycle consists of (1) filling an empty syringe with a washing solution while immersing [both the needle and] the base of the barrel into the washing solution, without fully removing the plunger, and (2) emptying the syringe into the waste beaker."
- »MiPNet19.14 SOP Hamilton microsyringes«
- communicated by »Baglivo Eleonora« and »Timon-Gomez Alba«

Mar-20 We

What if Alert: Error in titrations! appears in your O2 analysis template file?

The O2 analysis template recognizes discrepancies between the protocol selected in the first tab and the imported marks. Check the titration sequence and mark names against the protocol copied from the DLD file.
- »O₂ flux analysis«
- communicated by »Timon-Gomez Alba« and »Leo Elettra«

Mar-07 Th

Which medium can be used for the instrumental background test?

When using dithionite, the instrumental background test should be performed in mitochondrial respiration medium MiR05, MiR05-Kit or MiR06; or alternatively, in a strong alkaline phosphate buffer (>100 mM; >pH 8), to avoid side reactions that might interfere with the test.
- »MiPNet14.06_Instrumental_O2_background«
- communicated by »Schmitt Sabine« and »Timon-Gomez Alba«

Mar-04 Mo

Do you observe an unstable O₂ slope negative following TIP2k titrations during an instrumental O₂ background test?

Incorrect placement of TIP2k Filter Papers can lead to liquid accumulation and convective oxygen backdiffusion. Check for proper placement by confirming they are fully in contact with the stopper receptacle.
- »Instrumental O₂ background«, »Unstable O₂ slope negative«
- communicated by »Grings Mateus« and »Baglivo Eleonora«

Feb 2024

Feb-22 Th

What to do if the glass of the Oroboros chamber starts to become opaque?

"Turbidity on the glass wall may be caused by precipitated protein. Remove the chambers from the Oroboros and with the stirrers removed, immerse in a beaker filled with HCl (10 M) overnight under a hood."
-»MiPNet19.03_O2k-cleaning_and_ISS«
- communicated by »Cardoso Luiza« and »Leo Elettra«

Feb-08 Th

Can I use the same stock solutions for all reagents for different Oroboros chamber volumes?

"For some reagents, the concentration of the stock solution differs between the 2.0-mL and the 0.5-mL chamber (Oroboros sV-Module) to facilitate a precise volume titration."
-»MiPNet09.12 O2k-Titrations«
- communicated by »Timon-Gomez Alba« and »Leo Elettra«

Jan 2024

Jan-25 Th

How do I view and change the settings of a measurement/file in DatLab 8?

"Oroboros control (active) will always show the settings of the active recording [and allow changes], while Oroboros info (view) will show the settings of the recorded selected file."

-»MiPNet28.07 NextGen-O2k Series XB Manual«; »MiPNet28.08 Oroboros O2k Series J Manual«
- communicated by »Baglivo Eleonora« and »Grings Mateus«

Jan-11 Th

What are special features of the new SmartPOS (O2k-Series J and NextGen-O2k-Series XB)?

- "The electrical connections of the SmartPOS are protected thus there is no need to clean them."
- "The serial number of the SmartPOS is automatically recognized by the DatLab 8 software."

»MiPNet28.10 POS-service«
- communicated by »Grings Mateus« and »Baglivo Eleonora«

Jan-08 Mo

Do you need to export DatLab traces for your publication?

Save your graph as an editable picture using "Copy to clipboard" in DatLab 7.4 (.wmf) or "Save" in DatLab 8 (.svg).
- »Copy to clipboard in DatLab 7«, »Download DatLab 8«
- communicated by »Baglivo Eleonora« and »Grings Mateus«