Run DL-Protocol/Set O2 limit: Difference between revisions
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::::*'''DL-Protocol colors'''. In the process of running a DL-Protocol following the '''DL Protocol events''' ('''>> E:''' or '''<big>β</big> E:''') and even after | ::::*'''DL-Protocol colors'''. In the process of running a DL-Protocol, following the '''DL Protocol events''' ('''>> E:''' or '''<big>β</big> E:''') and even after when setting '''DL Protocol marks''' ('''>> M:''') in the [[#DL-Protocols-anchor5|list]] of the [[#DL-Protocol window|'DL-Protocol window']], events and marks appear in different colors according to the following rules: | ||
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! ColorΒ Β Β !! typeΒ !! indicator !! | ! ColorΒ Β Β !! typeΒ !! indicator !! Rules, description | ||
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|style="background:#FFFF00"| '''Yellow'''Β Β || eventsΒ || "'''>> E:'''" or "'''<big>β</big> E:'''" || indicates the next DL-Protocol event going to be completed | |style="background:#FFFF00"| '''Yellow'''Β Β || eventsΒ || "'''>> E:'''" or "'''<big>β</big> E:'''" || indicates the next DL-Protocol event going to be completed |
Revision as of 17:38, 26 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 [Β΅M] 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 [Β΅M]. 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 "β E:". Every mark or event is unique and must be set only once. However, there is an exception for events identified by "β", which can be set consecutively multiple times (multi-events), as it is necessary for titrating the uncoupler (U) in SUIT protocols.
- 'Edit DL-Protocol' window. In the listed events and marks, changes can be made in 'Final conc. [mM]', 'Titration vol. [Β΅L]' 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. [Β΅L]' 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 "β E:" (multi-events).
- Final conc. [mM] and Titration vol. [Β΅L]. 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 "β ") 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
- 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.
- DL-Protocol basics.
- The 'DL-Protocol window' shows the seqence of DL-Protocol events and DL-Protocol marks. For a better understanding, when running a DL-Protocol 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 but will be stored togehter with the DL-Protocol marks and events in the .DLD data file. It is possible to change a user-defined mark or user-defined event into DL-Protocol mark or DL-Protocol event, respectively.
- An HRFR experiment is primarily an event-driven process, thus, 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. However, DL-Protocol marks or any user-defined marks can be set at any time during the experimental run.
- DL-Protocol basics.
- DL-Protocol colors. In the process of running a DL-Protocol, following the DL Protocol events (>> E: or β 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 "β E:" indicates the next DL-Protocol event going to be completed Green event ">> E:" DL-Protocol event is completed and the position of the actual event or defined order of all events in the selected DL-Protocol has not been violated (e.g. wrong position, any missing events in the processed seuqence, ...) Aqua mulit event "β E:" DL-Protocol multi event is completed and position of the actual multi event or defined order of all events in the selected DL-Protocol has not been violated (e.g. wrong position, any missing events in the processed seuqence, ...). As intended, the multi event "β 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:" zzz Red marks, events ">> M:", ">> E:" or "β E:" White marks, events ">> M:", ">> E:" or "β E:"
- 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 [Β΅M] as specified.
- Titration volume, v [Β΅l]
- Chamber volume, V [Β΅l]
- Final concentration, cB [mM] or [Β΅M] as specified.
- cB [mM] = cB,in [mM] x v [Β΅l] / V [Β΅l]
- Example: Malate (M), cM,in = 800 mM stock; v = 5 Β΅l titration volume; V = 2,000 Β΅l chamber volume.
- cM [mM] = cM,in [mM] x v [Β΅l] / V [Β΅l] = 800 mM x 5 Β΅l / 2,000 Β΅l = 2 mM