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

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.

• 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'.

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. 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.
  • 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. 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 in action. 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.

• Marks in action 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. 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 following rules:

Color	type	indicator	Rules, description
Yellow	events	">> E:" or "∗ 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	"∗ E:"	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:"	The DL-Protocol mark occurs at the right position, after the associated DL-Protocol event.
Red	events	">> E:" or "∗ E:"	(i) 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.
	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 "∗ E:"	The DL-Protocol event is not set in the plot.
	marks	">> M:"	The DL-Protocol mark is not set in the plot.

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.

Event information window - Set event)

• 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 (>> E: xxx) 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: xxx) in the DL-Protocol window list. Proceed with the next yellow DL-Protocol event >> E: yyy at the according time point (after flux has stabilized or experiment specific timing). 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.

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