MiPNet17.05 O2k-Fluo LED2-Module

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MiPNet17.05 O2k-Fluo LED2-Module

Publications in the MiPMap
O2k-Manual
O2k-Fluo LED2-Module.

» Bioblast pdf

OROBOROS (2015-06-15) Mitochondr Physiol Network

Abstract: Fasching M, Gradl P, Gnaiger E (2015) O2k-Fluo LED2-Module. Mitochondr Physiol Network 17.05(08):1-6. »Versions


O2k-Manual: The O2k-Fluo LED2-Module is a modular extension of the O2k-Core (Series D upwards). A growing number of fluorescence markers enables determination of diverse mitochondrial processes in addition to oxygen consumption, including generation of H2O2, ATP production, mitochondrial membrane potential and Ca2+, extendable by user-specific applications.

» Product: O2k-Fluorometer, O2k-Fluo LED2-Module, O2k-Catalogue

HRR, Fluorometry O2k-Network Lab: AT_Innsbruck_OROBOROS


Labels: MiParea: Respiration, Instruments;methods  HRR: Oxygraph-2k, O2k-Fluorometer, O2k-Manual, O2k-Protocol Additional: O2k-MultiSensor 



OROBOROS support

O2k-technical support

This information is part of O2k-technical support and open innovation.


Contents


O2k-Manual: O2k-Fluo LED2-Module

Setup of the O2k-Fluo LED2-Module

  1. Setup of the O2k-Fluo LED2-Module
  2. Selecting a Fluorescence Sensor
  3. Mounting a Filter-Cap
  4. Connect Fluorescence-Sensor to O2k-Main unit
  5. Power on


LED-intensity and amplification

  1. LED-intensity
  2. Amplification
The light intensity of the LED (LED-intensity) and the signal amplification (Gain) can be adjusted in a wide range. The table suggests initial values, which can be optimised for specific applications.
  • The settings depend on the concentration of the fluorophore, which vary between different applications. Therefore, only recommendations for specific fluorophore concentrations are given. In the Amplex Ultrared assay the fluorophore is formed during the experiment.
  • The recommendations apply to experiments at 37 °C. The Fluorescence intensity increases strongly at lower temperatures. Then the light intensity is reduced to avoid off-scale signals.


Fluorophores

Application Sensor Filter set O2k output Light intensity (polarization voltage) - Note a Gain Comment
Amplex® UltraRedFluorescence-Sensor Green AmR Type B 100 - 500 1000 (at light intensity = 100)
TMRMFluorescence-Sensor Green AmR Type C 200 - 500 1000 at c(TMRM) = 2 µM
Safranin Fluorescence-Sensor Blue Saf Type C 100 - 200 for c(safranin)= 2 µM; 1000 at c(Mg Green) = 2 µM,
Magnesium greenFluorescence-Sensor Blue MgG / CaG Type B 100 - 300 1000 at c(Mg Green) = 2 µM
Calcium greenFluorescence-Sensor Blue MgG / CaG Type A and C 100 -300 1000 at c(Ca Green) = 2 µM
  • Note a: Set the polarization voltage [mV] for the amperometric channel (Amp) in the DatLab menu [O2k-MultiSensor \ O2k Control \ Amp polarisation voltage]. Divide the polarisation voltage [mV] by 100 to obtain the current [mA] through the LED. For simple operation instructions, it is sufficient to refer to the polarization settings selected in DatLab.


TMRM with the O2k-Fluo LED2-Module: Experiments with permeabilized HEK 293T cells by M. Hansl and G. Krumschnabel

HEK 293T cells
1.5 106 cells /ml
Experimental buffer: MiR05
Pyruvate: 5 mM; Malate: 2 mM; ADP: 2.5 mM; Succinate: 10 mM; CCCP (U) added in 0.5-1 µM steps; Rotenone: 1 µg/ml; Antimycin A: 2.5µM; TMRM: 2 µM final concentration
Experimental conditions:
2 ml chamber volume, Fluorescence-Sensor Green, Filter Set AmR, light intensity: Amp Polarization voltage = 500 , gain 1000, T = 37.0 °C


TMRM permHEK293T.png

Figure legend: Simultaneous measurement of respiration [upper panel; pmol/(s*ml)] and mitochondrial membrane potential (mtMP) (lower panel; arbitrary units) in permeabilized HEK 293T cells: TMRM was added to the O2k chamber with 2 ml experimental buffer in two 1 µl steps to obtain 2 µM TMRM and then 1.5 106 cells/ml were added. Next, cells were permeabilized by adding 10 µg/ml digitonin and this was followed by addition of pyruvate and malate to induce LEAK respiration. Then, 2.5 mM ADP was added to elicit CI-linked OXPHOS, then succinate was injected to obtain CI&II-linked OXPHOS, before titrating uncoupler CCCP (U) in 0.5-1 µM steps to induce fully uncoupled respiration and fully collapsed mtMP. It should be noted that in the permeabilized cells CCCP concentration required for complete collapse of mtMP was lower than that needed for maximum respiration. Finally, CI-inhibitor rotenone (Rot) and CIII-inhibitor antimycin A (Ama) were added to obtain residual oxygen consumption (ROX), which however did not further affect mtMP (further details: Talk:TMRM).


The fluorescence signal

  1. O2k signal: The O2k-Fluo LED2-Module is operated through the amperometric (Amp)-Channel of the O2k, with electric current (ampere [Amp]) as the primary signal.
  2. O2k output: type A, B, or C, or combinations.
Check Amp raw signal form Graph/Select Plots to display the fluorometric signal
Graph / Select plots‎
Graph layout: Three plots are available in DatLab based on the recorded signal: Amp Raw Signal, Amp Calibrated, and Amp Slope. These plots can be selected from the drop-down lines and displayed with their check boxes either on the Y1 or Y2 [Graph layout / Select Plots].


Amp Raw Signal displays the raw voltage (including amplification) as recorded by the O2k at a given gain setting.
Amp Calibrated is the signal after calibration with the parameters set in the O2k-MultiSensor Calibration window.
Amp slope is the time derivative of the calibrated signal, multiplied by 1000, in units [m(conc. Unit during calibration)/s], so if the signal was calibrated in µM [nmol/ml] the unit of the slope is pmol/(s ml). To obtain the slope of the raw signal check the appropriate box in the calibration window (DatLab 5.1.0.130 and above).
Graphs can be generated to display oxygen and fluorescence data, or several graphs can be added to display oxygen and fluorescence data separately. Layout templates are provided, which can be modified and saved as appropriate. All graph settings can be saved as user-defined layouts MiPNet12.07.


O2k-Fluorometry and the TIP2k

  • TiP2k: Our tests indicated that the fluorometric signal is not affected by the TIP2k needle inserted into the O2k-Chamber.
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