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__TOC__ == Additional resources == »[[MiPNet17.05 O2k-Fluo LED2-Module|O2k-Fluo Manual]] »[[O2k-Demo_experiments#O2k-Demo_O2k-MultiSensor |O2k-Demo experiments]] »[[Fluorescence-Sensor]] »{{MitoPedia methods |mitopedia method=Fluorometry }} »{{MitoPedia O2k and high-resolution respirometry |mitopedia O2k and high-resolution respirometry=O2k hardware }} [[File:Questions.jpg|left|40px]] <br /> <div class="toccolours mw-collapsible mw-collapsed"> ::: <span style="font-size:105%; color:##424242">'''» Keywords'''</span> <div class="mw-collapsible-content"> ::: '''Specific''' ::::» [[O2k-Fluorometer Series G]] ::::» [[Amplex UltraRed]] ::::» [[Calcium green]] ::::» [[Magnesium green]] ::::» [[Safranin]] ::::» [[TMRM]] ::::» [[Fluorescence-Control Unit]] ::::» [[Fluorescence-Sensor Blue]] ::::» [[Fluorescence-Sensor Green]] ::: '''General''' ::::» [[HRR]] ::::» [[mt-membrane potential]] ::::» [[O2k-Chamber]] ::::» [[O2k-Core]] ::::» [[O2k-Main Unit]] ::::» [[O2k signals and output#O2k output |O2k output]] ::::» [[O2k signals and output#Signal of the O2k-Core and add-on modules |O2k signal]] ::::» [[Oroboros O2k]] </div> </div> <br /> == O2k signal and output == ::::# [[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 (ampere [A]) as the primary signal. ::::# [[O2k signals and output#O2k output |O2k output]]: type A, B, or C, or combinations. == Scope of applications == :::: The scope of applications is defined by the excitation wavelengths of the Fluorescence-Sensor Green and Fluorescence-Sensor Blue and the selection of emission filters. :::: The O2k-Fluo LED2-Module is designed for measurements performed on samples placed in a stirred and temperature-controlled chamber. Fluorometric protocols based on measurements in cuvettes of various fluorometers can be used with the O2k-Fluo LED2-Module, and extended by simultaneous measurement of respiration and monitoring or regulation of oxygen concentration. The excitation and emission wavelengths of the fluorophore must be suitable for use with the O2k-Fluo LED2-Module. For examples see [[MiPNet17.05 O2k-Fluorescence LED2-Module#LED-intensity and amplification|LED-intensity and amplification]]. :::: The O2k-Fluo LED2-Module is not designed to fulfill the functions of fluorescence microscopes or flow cytometers. == O2k-Manual: O2k-Fluo LED2-Module (for O2k series D-G)== === 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 °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'' !! 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 µM |- | [[Safranin]] ||[[Fluorescence-Sensor Blue]]|| [[Filter Set Saf|Saf]] || [[O2k_signal_and_output| Type C]] || 100 - 200 || 1000 || at c(safranin) = 2 µM |- | [[Magnesium green]]||[[Fluorescence-Sensor Blue]]|| [[Filter Set MgG / CaG| MgG / CaG]] || [[O2k_signal_and_output| Type B]] || 100 - 500|| 1000 || at c(Mg Green) = 2 µM |- | [[Calcium green]]||[[Fluorescence-Sensor Blue]]|| [[Filter Set MgG / CaG| MgG / CaG]] || [[O2k_signal_and_output|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 === ::: '''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 µM steps; Rotenone: 1 µg/ml; Antimycin A: 2.5µM; TMRM: 2 µM 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 °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 µl steps to obtain 2 µM TMRM and then 1.5 10<sup>6</sup> 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 === :::# [[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 (ampere [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 plots ]]'''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 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, 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.== O2k-Fluorometry Workshops == ::::* [[Oroboros_Events#Next_O2k-Workshops|Oroboros O2k-Workshops]] [[Image:O2k-Publications.jpg|left|116px|link=O2k-Publications: Topics|O2k-Publications in the MiPMap]] <br /> <div class="toccolours mw-collapsible mw-collapsed"> ::: <span style="font-size:105%; color:##424242">'''» References'''</span> <div class="mw-collapsible-content"> '''»[[O2k-Publications: Topics]]''' '''''Sort in ascending/descending order by a click on one of the small symbols in squares below'''''. Default sorting: chronological. Empty fields appear first in ascending order. {{#ask:[[Category:Publications]] [[Instrument and method::Oxygraph-2k]] [[Instrument and method::O2k-Fluorometer]] |?Was published in year |?Has title |?Mammal and model=Organism |?Tissue and cell=Tissue;cell |format=broadtable |limit=5000 |offset=0 |sort=Was published in year |order=descending }} </div> </div> <br /> [[Image:O2k-Publications.png|left|115px|link=O2k-Publications: Topics|O2k-Publications in the MiPMap]] <br /> <div class="toccolours mw-collapsible mw-collapsed"> ::: <span style="font-size:105%; color:##424242">'''» O2k-Publications: O2k-Fluorometry - Abstracts'''</span> <div class="mw-collapsible-content"> '''''Sort in ascending/descending order by a click on one of the small symbols in squares below'''''. {{#ask:[[Category:Abstracts]] [[Instrument and method::Oxygraph-2k]] [[Instrument and method::O2k-Fluorometer]] |?Was submitted in year |?Has title |?Mammal and model=Organism |?Tissue and cell=Tissue;cell |format=broadtable |limit=5000 |offset=0 |sort=Was submitted in year |order=descending }} </div> </div> <br /> == Next step == ::::* [[O2k-FluoRespirometer]] ::::* [[O2k-Innovation description|O2k-Innovation]]
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