Cookies help us deliver our services. By using our services, you agree to our use of cookies. More information

Difference between revisions of "MiPNet17.05 O2k-Fluo LED2-Module"

From Bioblast
(6 intermediate revisions by 3 users not shown)
Line 1: Line 1:
{{OROBOROS header page name}}
{{Template:OROBOROS support page name}}
{{Publication
{{Publication
|title=[[Image:O2k-Manual.jpg|right|70px|link=O2k-Manual|O2k-Manual]] O2k-Fluo LED2-Module.
|title=[[Image:O2k-Manual.jpg|right|70px|link=O2k-Manual|O2k-Manual]] O2k-Fluo LED2-Module.
Line 6: Line 6:
|year=2016-08-09
|year=2016-08-09
|journal=Mitochondr Physiol Network
|journal=Mitochondr Physiol Network
|abstract='''Fasching M, Gradl P, Gnaiger E (2016) O2k-Fluo LED2-Module. Mitochondr Physiol Network 17.05(09):1-6.'''
|abstract=Fasching M, Gradl P, Gnaiger E (2016) O2k-Fluo LED2-Module. Mitochondr Physiol Network 17.05(09):1-6. Β 
Β 
{{MiPNet pdf page linking to MitoPedia}}
Β 
'''O2k-Manual:''' The O2k-Fluo LED2-Module is a modular extension of the O2k-Core (O2k-Series D to G). A growing number of fluorescence markers enables determination of diverse mitochondrial processes in addition to oxygen consumption, including generation of H<sub>2</sub>O<sub>2</sub>, ATP production, mitochondrial membrane potential and Ca<sup>2+</sup>, extendable by user-specific applications.
:Β» Product: [[O2k-Fluo LED2-Module]], [[Oroboros O2k-Catalogue |O2k-Catalogue]]
:Β» Product: [[O2k-Fluo LED2-Module]], [[Oroboros O2k-Catalogue |O2k-Catalogue]]
|keywords=[[HRR]], [[Fluorometry]]
|mipnetlab=AT_Innsbruck_Oroboros
|mipnetlab=AT_Innsbruck_Oroboros
}}
}}
Line 17: Line 14:
|area=Respiration, Instruments;methods
|area=Respiration, Instruments;methods
|instruments=Oxygraph-2k, O2k-Fluorometer, O2k-Manual, O2k-Protocol
|instruments=Oxygraph-2k, O2k-Fluorometer, O2k-Manual, O2k-Protocol
|additional=O2k-MultiSensor
|additional=O2k-MultiSensor, DatLab, Archive
}}
}}
* Contribution to K-Regio project ''MitoCom Tyrol'', funded in part by the Tyrolian Government and the European Regional Development Fund (ERDF).&nbsp;&nbsp;&nbsp;Β  Β  >> [[MitoCom_O2k-Fluorometer|''MitoCom O2k-Fluorometer'']]
{{Template:Technical support integrated}}
__TOC__
== O2k-Manual: O2k-Fluo LED2-Module ==
=== Setup of the O2k-Fluo LED2-Module ===
:::# [[Fluorescence-Control_Unit#Setup_of_the_O2k-Fluorescence_LED2-Module|Setup of the O2k-Fluo LED2-Module]]
:::# [[Fluorescence-Sensor#Select_the_Fluorescence-Sensors|Selecting a Fluorescence Sensor]]'''
:::# [[Filter-Cap#Mounting_a_Filter-Cap|Mounting a Filter-Cap]]
:::# [[Fluorescence-Sensor#Connect_Fluorescence-Sensor_to_O2k|Connect Fluorescence-Sensor to O2k-Main unit]]
:::# [[Fluorescence-Control_Unit#Power_on|Power on]]
=== LED-intensity and amplification ===
:::# [[Fluorescence-Control_Unit#Control_of_LED-intensity|LED-intensity]]
:::# [[Fluorescence-Control_Unit#Gain|Amplification]]
::::The light intensity of the LED ([[Fluorescence-Control_Unit#Control_of_LED-intensity|LED-intensity]]) and the signal amplification ([[Fluorescence-Control_Unit#Gain|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 Β°C. The Fluorescence intensity increases strongly at lower temperatures. Then the light intensity is reduced to avoid off-scale signals.
== Fluorophores ==
{| class="wikitable"
|-
! Application !!Sensor!! Filter set !! O2k output !! Light intensity (polarization voltage) - ''Note a''Β  !! Gain !! Comment
|-
| [[Amplex UltraRed]]||[[Fluorescence-Sensor Green]] || [[ Filter Set AmR| AmR]] || [[O2k_signal_and_output|Type B]] || 100 - 500 || 1000 (at light intensity = 100) ||
|-
| [[TMRM]]||[[Fluorescence-Sensor Green]] || [[ Filter Set AmR| AmR]] || [[O2k_signal_and_output|Type C]] || 200 - 500|| 1000 || at c(TMRM) = 2 Β΅M
|-
| [[Safranin]] ||[[Fluorescence-Sensor Blue]]|| [[Filter Set Saf|Saf]] || [[O2k_signal_and_output| Type C]] || 100 - 200 for c(safranin)= 2 Β΅M; || 1000 || at c(Mg Green) = 2 Β΅M,
|-
| [[Magnesium green]]||[[Fluorescence-Sensor Blue]]|| [[Filter Set MgG / CaG| MgG / CaG]] || [[O2k_signal_and_output| Type B]] || 100 - 300|| 1000 || at c(Mg Green) = 2 Β΅M
|-
| [[Calcium green]]||[[Fluorescence-Sensor Blue]]|| [[Filter Set MgG / CaG| MgG / CaG]] || [[O2k_signal_and_output|Type A and C]] ||100 -300||Β  1000 ||at c(Ca Green) = 2 Β΅M
|}
::::* ''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 10<sup>6</sup> 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 Β΅M steps; Rotenone: 1 Β΅g/ml; Antimycin A: 2.5Β΅M; TMRM: 2 Β΅M final concentration
:::: '''Experimental conditions:'''
:::: 2 ml chamber volume, [[Fluorescence-Sensor_Green|Fluorescence-Sensor Green]], [[Filter_Set_AmR|Filter Set AmR]], light intensity: Amp Polarization voltage = 500 , gain 1000, T = 37.0 Β°C
[[Image:TMRM_permHEK293T.png|600px]]
:::: '''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 Β΅l steps to obtain 2 Β΅M TMRM and then 1.5 10<sup>6</sup> cells/ml were added. Next, cells were permeabilized by adding 10 Β΅g/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 Β΅M 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 signalΒ  ===
:::# [[O2k signals and output#Signal of the O2k-Core and add-on modules |O2k signal]]: The [[O2k-Fluo LED2-Module]] is operated through the amperometric (Amp)-Channel of the O2k, with electric current (ampereΒ  [Amp]) as the primary signal.
:::# [[O2k signals and output#O2k output |O2k output]]: type A, B, or C, or combinations.
[[Image:DL7graph_select_plots_amp.jpg|thumb|250px|alt=Check Amp raw signal form Graph/Select Plots to display the fluorometric signal| Graph / Select plotsβ€Ž ]]'''Graph layout''': Three plots are available in DatLab based on the recorded signal: '''Amp Raw Signal''', '''Amp Calibrated''', and '''Amp Slope'''<nowiki>. 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].</nowiki>
:::: '''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 [mV(conc. Unit during calibration)/s], so if the signal was calibrated in Β΅M [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, see [[MiPNet19.01C DatLab Guide]].
== O2k-Fluorometry and the TIP2k ==
::::* [[Titration-Injection microPump |TiP2k]]: Our tests indicated that the fluorometric signal is not affected by the TIP2k needle inserted into the O2k-Chamber.

Revision as of 21:59, 13 February 2020


                  


O2k-Open Support

MiPNet17.05 O2k-Fluo LED2-Module


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

Β» Bioblast pdf Β»Versions

Oroboros (2016-08-09) Mitochondr Physiol Network

Abstract: Fasching M, Gradl P, Gnaiger E (2016) O2k-Fluo LED2-Module. Mitochondr Physiol Network 17.05(09):1-6.

O2k-technical support and open innovation
Open the pdf document above.
Β» Current O2k-series: NextGen-O2k Series XB and O2k Series J
Β» Current software versions DatLab 8.0: MitoPedia: DatLab
Β» Product: O2k-Fluo LED2-Module, O2k-Catalogue


β€’ O2k-Network Lab: AT_Innsbruck_Oroboros


Labels: MiParea: Respiration, Instruments;methods 





HRR: Oxygraph-2k, O2k-Fluorometer, O2k-Manual, O2k-Protocol 

O2k-MultiSensor, DatLab, Archive