Run DL-Protocol/Set O2 limit

high-resolution terminology - matching measurements at high-resolution

Run DL-Protocol/Set O2 limit

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 [µM] 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, DLPU

MitoPedia O2k and high-resolution respirometry: DatLab

Protocol menu

File:Protocol-menu.jpg

Protocol menu

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 For chamber A (upper frame) a specific protocol is selected, showing the name ("RP1mt"), the hyperlink to the Biobast in blue and additional information. Furthermore, the 'Lower O2 limit [µM]' alert is checked. In chamber B (lower frame) no DL-protocol is selected.

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 Oroboros, '.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.

O2-limit warning button

Lower O2 limit [µM]. When checked, an automatic warning notifies the user when the actual oxygen level falls below the specified value for the according 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 is 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

DL-Protocol window in DatLab, highlighted by a red box in the image. The DL-Protocol windows for chamber A (up) and chamber B (down) are shown with different DL-protocols as indicated by the DL-protocol name in the name field (1, red oval)

The DL-Protocol window appears to the very right for every chamber. It consists 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.

'Event Information window' SET and EDIT

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 "∗ E:". Every mark or event is unique and must be set only once. However, there is an exception for events identified by "∗", 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 and mouse action'. Further, the marks and events can be left- or right clicked to open specific windows - dependend on the color-code - for editing or setting or simply viewing information (see the list in 'DL-Protocol colors and mouse action'):

- 'Event Information window' SET and EDIT. This 'Event Information window' only shows up when DatLab 7 is connected to the O2k and a DL-Protocol event in white, yellow or aqua is left-clicked. Beside the possibility to edit information or to even select a DL-Protocol event, it's main purpose ist to set the event in the pot with the according name and the specific time.

- 'Event Information window' only EDIT. This 'Event Information window' shows up when DatLab 7 is connected or disconnected and a DL-Protocol event in green, aqua or red is right-clicked. It only allows to edit the DL-Protocol event or even change to another DL-Protocol from the list. not set the event. Ther is no option to set the event.

- 'Event Information window' MINIMAL . This 'Event Information window' shows up when DatLab 7 is connected or disconnected and a DL-Protocol event in white or yellow. It only shows the DL-Protocol event specific information, there is no option to edit or set the event.

- 'Mark Information window' EDIT. This 'Mark Information window' shows up when DatLab 7 is connected or disconnected and a DL-Protocol mark in light-green or red is right-clicked (no left-click effect for DL-Protocol marks!). It allows to edit the DL-Protocol mark or even to change to another one from the list.

- 'Mark Information window' MINIMAL. This 'Mark Information window' shows up when DatLab 7 is connected or disconnected and a DL-Protocol mark in white is right-clicked (no left-click effect for DL-Protocol marks!). It only shows the DL-Protocol mark specific information, there is no option to edit or set the event.

Edit DL-protocol window ...

'Edit DL-Protocol' window. In the listed events and marks, changes can be made in 'Final conc. [mM]', 'Titration vol. [µL]' 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 available.

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 "∗ E:" (multi-events).
- Final conc. [mM] and Titration vol. [µL]. 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 , whereupon the indicator (">>" or "∗ ") for the event changes. For marks, a set checkbox is of no relevance.

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.

DL-Protocol principles

Events and marks basics.
- The 'DL-Protocol window' shows the sequence 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 together with the DL-Protocol marks and events in the .DLD data file. Extra user-defined events are of importance for unpredictable occurrences e.g. "Open" and "Close" events, when a chamber has to be re-oxygenised.
- It is possible to edit/change a user-defined mark or user-defined event into DL-Protocol mark or DL-Protocol event, respectively.
- 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).

Rules.
- Correct completion. 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 ("∗ E:"), which is allowed to be set successively multiple times before proceeding with the next DL-Protocol event in the DL-Protocol sequence. Color-coding of events and marks helps to keep track of the correct process through the DL-Protocol, see 'DL-Protocol colors'.
- Events first. 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. As stated above, a user-defined event can be set at any time inbetween the DL-Protocol events. If needed, DL-Protocol marks or any user-defined marks are allowed to be set at any time during the experimental run.
- Marks second 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 preceding DL-Protocol event, as determined in DL-Protocol sequence. Only when set behind the corresponding DL-Protocol event, The DL-Protocol mark is correctly set and the 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 preceding DL-Protocol event. When setting a second or any further mark in a state (behind a DL-Protocol event), this mark will be recognised as a user-defined mark, automatically named with a single letter (alphabetically, 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 colors and mouse action In the process of running a DL-Protocol, following the DL Protocol events (>> E: or ∗ 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 rules in the table below. Depending on the color, different 'Information' windows appear Please note that there is no left-click mouse action when DatLab 7 is not connected to the O2k.

Color	type	indicator	left-click (only when connected)	right click (connected and offline)	Rules, description
White	events	">> E:" or "∗ E:"	SET and EDIT 'Event information window'	MINIMAL 'Event information window'	The DL-Protocol event is not set yet and no rules have been violated, according to the DL-Protocol.
White	marks	">> M:"	no effect	MINIMAL 'Mark information window'	The DL-Protocol mark is not set yet and no rules have been violated, according to the DL-Protocol.
Yellow	events	">> E:" or "∗ E:"	SET and EDIT 'Event information window'	MINIMAL 'Event information window'	The DL-Protocol event is not set yet. Indicates the next DL-Protocol event going to be set according to the DL-Protocol.
Green	event	">> E:"	no effect	only EDIT 'Event information window'	The DL-Protocol event is set/completed in the correct position according to the DL-Protocol.
Aqua	mulit event	"∗ E:"	SET and EDIT 'Event information window'	only EDIT 'Event information window'	The DL-Protocol multi event is set/completed in the correct position according to the DL-Protocol. As intended, a multi event "∗ 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:"	no effect	EDIT 'Mark information window'	The DL-Protocol mark is set/occurs at the right position, after the associated DL-Protocol event.
Red	events	">> E:" or "∗ E:"	only EDIT 'Event information window'	only EDIT 'Event information window'	(i) The DL-Protocol event is set but is wrong for the following reasons: The DL-Protocol event occurs in the wrong position e.g. a later on DL-Protocol event from the DL-Protocol sequence 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 sequence - can lead to a sequence of red events. (iii) The "">> E:" DL-Protocol event (not a multi-event!) occurs multiple times in the plot.
Red	marks	">> M:"	no effect	EDIT 'Mark information window'	The DL-Protocol mark is set but wrong for the following reasons: (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.

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 displayed 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.
- Considering the experimental timing (e.g. flux has stabilized), left-klick onto the first DL Protocol event in yellow (>> E: 1xxx) of the DL Protocol and the Event information window will open to set the DL-Protocol event in the plot. When the DL-Protocol event is set correctly according to the protocol sequence it turns into green (>> E: 1xxx) in the DL-Protocol window list.
- Proceed with the next yellow DL-Protocol event >> E: 2xxx at the according time point (after flux has stabilized or experiment specific timing).
- Please note: White and yellow color-coded events are not yet set, yellow only indicates (logically) for the next event in the DL-Protocol to be set. Thus, left cklicking any white DL-Protocol event in the list will also open the 'Event information window' to set the event. However, setting the white DL-Protocol event will lead to a violation of the DL-Protoccol sequence and this incorrect DL-Protocol event will appear in red. This problem is solved by editing the wronlgy set DL-Protocol event (right-click in the 'DL-Protocol window' list to open the 'Event information window' or left cklicking in the plot) and delete it from the plot. Then just proceed with the yellow DL-Protocol event.
- When a multi-event is encountered the first time to be set it appears in yellow (∗ E: xxx *). After that it appears in auqua (∗ E: xxx *) and can be set multiple times, according to the experimental needs. Don't be confused that the next DL-Protocol event in the list (after the multievent) is already displayd in yellow!
- User-defined envents can be set during the run at any time point e.g. for "O" (open) and "C" (close) events when re-oxygenation o the chamber is necessary. Therfore chose menu 'Experiment' -> 'Add event' (press F4) or STRG-left-click in plot. By default, a user-defined envents will always be named "Event" in the plot an will not appear in the DL-Protocol list.

Titrations in SUIT protocols

Stock concentration of substance B in the titration syringe, c_B,in [mM] or [µM] as specified.
Titration volume, v [µl]
Chamber volume, V [µl]
Final concentration, c_B [mM] or [µM] as specified.

c_B [mM] = c_B,in [mM] x v [µl] / V [µl]

» MiPNet09.12 O2k-Titrations

Example: Malate (M), c_M,in = 800 mM stock; v = 5 µl titration volume; V = 2,000 µl chamber volume.

c_M [mM] = c_M,in [mM] x v [µl] / V [µl] = 800 mM x 5 µl / 2,000 µl = 2 mM