Run DL-Protocol/Set O2 limit: Difference between revisions
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::::*'''DL-Protocol basics'''. | ::::*'''DL-Protocol basics'''. | ||
::::**The [[#DL-Protocol window|'DL-Protocol window']] shows the seqence of '''DL-Protocol events''' and '''DL-Protocol marks''' that have to be set to complete an experiment. Besides, it is possible to set '''user-defined marks''' and '''user-defined events''' (menu 'Experiment' -> 'Add event' or F4 or STRG-left-click in plot), which will not affect the DL-Protocol | ::::**The [[#DL-Protocol window|'DL-Protocol window']] shows the seqence of '''DL-Protocol events''' and '''DL-Protocol marks''' that have to be set to complete an experiment. Besides, it is possible to set '''user-defined marks''' and '''user-defined events''' (menu 'Experiment' -> 'Add event' or F4 or STRG-left-click in plot), which will not affect the DL-Protocol and will be stored togehter with the DL-Protocol marks and events in the .DLD data file. Extra '''user-defined events''' are of importance for unpredictable occurences e.g. when a chamber has to be re-oxygenised, requiring 'open' and 'close' events. It is possible to edit/change a '''user-defined mark''' or '''user-defined event''' into '''DL-Protocol mark''' or '''DL-Protocol event''', respectively. | ||
::::**For the correct completion of a DL-Protocol, every single '''DL-Protocol mark''' ("'''>> M:'''") or '''DL-Protocol event''' ("'''>> E:'''") must be set only once in the right order. The exception is a '''DL-Protocol multi-event''' ("'''<big>โ</big> E:'''"), which is allowed to be set successively multiple times before proceeding with the next DL-Protocol event in the DL-Protocol sequence. | ::::**For the correct completion of a DL-Protocol, every single '''DL-Protocol mark''' ("'''>> M:'''") or '''DL-Protocol event''' ("'''>> E:'''") must be set only once in the right order. The exception is a '''DL-Protocol multi-event''' ("'''<big>โ</big> E:'''"), which is allowed to be set successively multiple times before proceeding with the next DL-Protocol event in the DL-Protocol sequence. | ||
::::**An HRFR experiment is primarily an '''event'''-driven process: '''DL-Protocol events''' MUST be set; '''DL-Protocol marks''' DON'T NEED to be regarded at first, when running the DL-Protocol. Hence, for practicability, the '''DL-Protocol marks''' can be hidden in the [[#DL-Protocols-anchor5|list]] ([[#DL-Protocol window|'DL-Protocol window']]: [[File:Hide_marks.png|60px]]) and the user only has to focus on the sequence of the '''DL-Protocol events'''. Still, '''DL-Protocol marks''' or any '''user-defined marks''' can be set at any time during the experimental run. | ::::**An HRFR experiment is primarily an '''event'''-driven process: '''DL-Protocol events''' MUST be set; '''DL-Protocol marks''' DON'T NEED to be regarded at first, when running the DL-Protocol. Hence, for practicability, the '''DL-Protocol marks''' can be hidden in the [[#DL-Protocols-anchor5|list]] ([[#DL-Protocol window|'DL-Protocol window']]: [[File:Hide_marks.png|60px]]) and the user only has to focus on the sequence of the '''DL-Protocol events'''. Still, '''DL-Protocol marks''' or any '''user-defined marks''' can be set at any time during the experimental run. |
Revision as of 13:38, 28 July 2017
Description
DL-Protocols can be selected in DatLab 7 in the pull-down menu 'Protocol': Set DL-Protocol / O2 limit. A DL-Protocol defines the sequence of Events and Marks. Instrumental DL-Protocols are used for calibrations and instrumental quality control, typically without experimental sample in the incubation medium. DL-Protocols for substrate-uncoupler-inhibitor titrations (see MitoPedia: SUIT) proceed stepwise to activate a sequence of coupling control states and pathway control states. A specific SUIT protocol can be assigned to O2k-chamber A or B or both. The Titration-Injection-microPump TIP2k can be programmed for automatic control of titration steps in a DL-Protocol. A collection of evaluated and tested standard DL-Protocols are provided. These can be edited and saved under 'Lab protocols' or under the User logged into DatLab. A Lower O2 limit [ยตM] can be defined for each chamber, to trigger an automatic warning when the experimental O2 concentration declines below this limit as a WARNING to remind the user that re-oxygenation of the chamber is required.
Abbreviation: DLP
MitoPedia O2k and high-resolution respirometry:
DatLab
- Show DL-Protocol can be check-marked () to hide or show the DL-Protocol window. Alternatively, the button (upper left) in the DL-Protocol window can be used to hide or show the window. When DatLab is started for data recording, an empty DL-Protocol window shows up because no DL-protocol is specified at this moment.
- Synchronous DL-Protocol events. This option is only available when the same DL-Protocol is selected (in 'Set DL-Protocol / O2 limit') for chamber A and B (protocol names are displayed in the name field of the DL-Protocol window). When checked , the user only needs to follow one protocol sequence of either chamber A or B. For example, when an event is set for chamber A, it will also be automatically set for chamber B - the 'Event Information' window indicates "Set event in both chambers" in bold letters.
- Set DL-Protocol / O2 limit. In the 'Set DL-Protocol / O2 limit' window a DL-Protocol can be assigned to specific chamber.
- Buttons. DL-protocols (either a .DLP or .DLPU file) can be selected () for chamber A and B. '.DLP' is the original file format for DL-Protocols provided by Orobors, '.DLPU' files are user-modified versions (see A: Export DL-Protocol and 'Edit DL-Protocol' window) of a specific DL-Protocols ('.DLP' file). Only when a DL-Protocol is initially selected, the button ist active and enables to automatically open the DL-Protocol file in another DatLab instance to see a DL-Protocol specific example data plot with properly set marks and events. Clicking the button removes the selected DL-Protocol.
- Additional information. When a DL-protocol is selected for chamber A or B further text information is provided in the chamber specific field: (1) protocol name, (2) a hyperlink (in blue, underlined) to the protocol in the Bioblast and (3) additional information and short description for the DL-Protocol.
- Lower O2 limit [ยตM]. When checked, an automatic warning notifies the user when the actual oxygne leves falls below the specified value for the accoring chamber: A red flashing "WARNING" button in the status line (DatLab window bottom, right) appears and a "WARNING" event is set in the plot. To reset the actual warning status (to notify the next possible fall-below), the red flashing "WARNING" button has to be clicked. First, a 'Warning window' opens to show all warnings during the experiment. After closing the window, the reset ist completed.
- A: Export DL-Protocol. The user can save changes in a DL-Protocol and export an user-modified DL-Protocol (.DLPU) for reuse. To make changes in a DL-Protocol see: 'Edit DL-Protocol' window
- B: Export DL-Protocol. applies for chamber B as described above in 'A: Export DL-Protocol'.
DL-Protocol window
- The DL-Protocol window appears to the very right for every chamber. It consisits of three buttons at the top, a name field and a list to display events and marks.
- Buttons. The button is used to hide/show the DL-Protocol window (similar to 'Show DL-Protocol' in the 'Protocol' menu). Clicking the / button toggles between hiding or showing the marks in the list. When the marks in the DL-Protocol are hidden the user only needs to focus on the sequence of events, facilitating the experimental procedure.The button opens the 'Edit DL-Protocol' window to make changes in events and marks in the DL-Protocol.
- Information. Below the buttons, the 'name field' automatically displays the name of the loaded protocol.
- List. The list in the DL-Protocol window shows the sequence of events and marks. The numbers in the event and mark names corresponds to the defined sequence in the DL-Protocol. Mark names are preceded by ">> M:", events are indicated by ">> E:" or "โ E:". Every mark or event is unique and must be set only once. However, there is an exception for events identified by "โ", which can be set consecutively multiple times (multi-events), as it is necessary for titrating the uncoupler (U) in SUIT protocols. The color-code for DL-Protocol marks and events in the list are described below in 'DL-Protocol colors'.
- 'Edit DL-Protocol' window. In the listed events and marks, changes can be made in 'Final conc. [mM]', 'Titration vol. [ยตL]' and 'Multi' but not in 'Type', 'Name' and 'Definition/State'. Further changes are accepted in 'Comment' and 'DL-Protocol description' at the bottom of the window.
- Information in the window head. The active plot is indicated in the according name field (window, top). A second name field displays the DL-Protocol name, furthermore, a hyperlink (blue, underlined) to the biobast is avialable.
- 'Edit DL-Protocol' window. In the listed events and marks, changes can be made in 'Final conc. [mM]', 'Titration vol. [ยตL]' and 'Multi' but not in 'Type', 'Name' and 'Definition/State'. Further changes are accepted in 'Comment' and 'DL-Protocol description' at the bottom of the window.
- List. In the list of the window, events are always displayed, however, only the marks (if available) for the active plot (O2 or O2 flux) are shown.
- Type and Name. Similar to the DL-Protocol window, mark names are preceded by ">> M:", events are indicated by ">> E:" or "โ E:" (multi-events).
- Final conc. [mM] and Titration vol. [ยตL]. The fields contain the according values for an event or mark and can be changed by the user.
- Multi. To toggle an events status from or to the status 'multi-event' ,the checkbox in the column has to be set or unchecked , wherupon the indicator (">>" or "โ ") for the event changes. For marks, a set checkbox is of no relevance.
- List. In the list of the window, events are always displayed, however, only the marks (if available) for the active plot (O2 or O2 flux) are shown.
- Comment. This field contains specific information for an event or mark and can be changed/edited by the user.
- DL-Protocol description. This field contains general, DL-Protocol specific information and can be changed/edited by the user.
- Changes can be accepted or rejected . Finally, changes in the DL-Protocol can be saved/exported to generate a user modified DL-Protocol (.DLPU), see A: Export DL-Protocol.
Running a DL-Protocol
- DL-Protocol basics.
- The 'DL-Protocol window' shows the seqence of DL-Protocol events and DL-Protocol marks that have to be set to complete an experiment. Besides, it is possible to set user-defined marks and user-defined events (menu 'Experiment' -> 'Add event' or F4 or STRG-left-click in plot), which will not affect the DL-Protocol and will be stored togehter with the DL-Protocol marks and events in the .DLD data file. Extra user-defined events are of importance for unpredictable occurences e.g. when a chamber has to be re-oxygenised, requiring 'open' and 'close' events. It is possible to edit/change a user-defined mark or user-defined event into DL-Protocol mark or DL-Protocol event, respectively.
- For the correct completion of a DL-Protocol, every single DL-Protocol mark (">> M:") or DL-Protocol event (">> E:") must be set only once in the right order. The exception is a DL-Protocol multi-event ("โ E:"), which is allowed to be set successively multiple times before proceeding with the next DL-Protocol event in the DL-Protocol sequence.
- An HRFR experiment is primarily an event-driven process: DL-Protocol events MUST be set; DL-Protocol marks DON'T NEED to be regarded at first, when running the DL-Protocol. Hence, for practicability, the DL-Protocol marks can be hidden in the list ('DL-Protocol window': ) and the user only has to focus on the sequence of the DL-Protocol events. Still, DL-Protocol marks or any user-defined marks can be set at any time during the experimental run.
- In most instances, a DL-Protocol event is a titration step of a specific substance according to the DL Protocol. Thereby, the DL-Protocol event demarcates a specific 'state' (in particular a respiratory state, depending on the titrated substance) until the next DL-Protocol event. Within a 'states' a DL-Protocol mark is set to read out the state-specific oxygen flux (or any other signal such as oxygen or fluorescence - has to be defined in the DL-Protocol).
- For practicability, it is recommended to set the DL-Protocol marks after the experimental procedure has been completed. By definition, a DL-Protocol mark is necessarily linked to a preceeding DL-Protocol event, as determined in DL-Protocol sequence. Only when set behind the corresponding DL-Protocol event, The DL-Protocol mark name is automatically assigned. With DL-Protocol events set in a plot, the DL-Protocol marks can be set in any order, because the automatic mark naming is based on the preceeding DL-Protocol event. When setting a second or any further mark in a state (behind a DL-Protocol event), this mark will be recoginsed as a user-defined mark, automatically named with a single letter (alphabatically, starting with 'a'). The same is true, when a mark is set in the plot behind an DL-Protocol event, where no DL-Protocol mark is defined in the DL-Protocol.
- DL-Protocol basics.
- DL-Protocol colors. In the process of running a DL-Protocol, following the DL Protocol events (>> E: or โ E:) and even after when setting DL Protocol marks (>> M:) in the list of the 'DL-Protocol window', events and marks appear in different colors according to the following rules:
Color type indicator Rules, description Yellow events ">> E:" or "โ E:" Indicates the next DL-Protocol event going to be set according to the DL-Protocol Green event ">> E:" The DL-Protocol event is set/completed in the correct position according to the DL-Protocol. Aqua mulit event "โ E:" The DL-Protocol multi event is set/completed in the correct position according to the DL-Protocol. As intended, a multi event "โ E: xxx *"(in auqua) can be consecutively set/clicked multiple times before processing with the next DL-Protocol event (in yellow) in the DL-Protocol.
Light green mark ">> M:" The DL-Protocol mark has occurs at the right position, after the associated DL-Protocol event. Red events ">> E:" or "โ E:" (i) The DL-Protocol event occurs in the wrong position e.g. a later on DL-Protocol event from the DL-Protocol seuqence has been set too early. (ii) A scheduled DL-Protocol event is missing in(has been left out) or has been deleted from the processed seuqence - can lead to a sequence of red events. (iii) The "">> E:" DL-Protocol event (not a multi-event!) occurs multiple times in the plot. marks ">> M:" (i) The DL-Protocol mark occurs in the wrong place according to the DL-Protocol. (ii) The DL-Protocol mark occurs more than once in the plot. (iii) According tot the rules, a DL-Protocol mark is always necessarily linked to a preceding DL-Protocol event. When this particular DL-Protocol event is coded red, also the associated DL-Protocol mark is considered wrong. White events ">> E:" or "โ E:" The DL-Protocol event is not set in the plot. marks ">> M:" The DL-Protocol mark is not set in the plot.
- Start. Hardware and Software must be setup properly (refer to the O2k-FluoRespirometer manual). DatLab 7 has to be started, connected ('Connect to O2k') and all instrumental- and experimental parameters have to be set.
- Loading a DL-Protocol. A DL-Protocol (either .DLP or DLPU) can be loaded for each chamber in 'Set DL-Protocol / O2 limit'. When the same DL-Protocol is loaded for both chambers, the 'Synchronous DL-Protocol events' mode of operation is available. The loaded DL-protocols for each chamber are diplayed in the 'DL-Protocol window' to the right in DatLab 7.
- Running a DL-Protocol. Once the DL-Protocol is loaded, the protocol layout will be displayed on the right side in the 'DL-Protocol window'. The experiment can now be started following the DL Protocol event sequence. For this purpose, left-klick onto the first DL Protocol event (in yellow) of the DL Protocol and the Event information window will open.
Titrations in SUIT protocols
- Stock concentration of substance B in the titration syringe, cB,in [mM] or [ยตM] as specified.
- Titration volume, v [ยตl]
- Chamber volume, V [ยตl]
- Final concentration, cB [mM] or [ยตM] as specified.
- cB [mM] = cB,in [mM] x v [ยตl] / V [ยตl]
- Example: Malate (M), cM,in = 800 mM stock; v = 5 ยตl titration volume; V = 2,000 ยตl chamber volume.
- cM [mM] = cM,in [mM] x v [ยตl] / V [ยตl] = 800 mM x 5 ยตl / 2,000 ยตl = 2 mM