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 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.

O2-limit warning button

Lower O2 limit [µM]. 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

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 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 "∗ 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 avialable.

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 , wherupon 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.

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 but 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. 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.
- 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. As the name and the DL-protocol sequence indicate, A DL-Protocol mark has to be set in a state at the right position: only behind the corresponding DL-Protocol event BUT before the next DL-Protocol event. Only then, facilitated by the DL-Protocol, 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 only based on a preceding 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 ture, when a mark is set in the plot after an DL-Protocol event where no DL-Protocol mark is defined.

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
Green	event	">> E:"	The 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.
Aqua	mulit event	"∗ E:"	The DL-Protocol multi event is completed and the position of the actual multi event or the 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, 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 has been set at the right position, after the associated DL-Protocol event.
Red	events	">> E:" or "∗ E:"	(i) A later on DL-Protocol event from the DL-Protocol seuqence has been set too early or the DL-Protocol event has been set at the wrong position. (ii) A scheduled DL-Protocol event is missing (has been left out) in or has been deleted from the processed seuqence - can lead to a sequence of red events. (iii) A "">> E:" DL-Protocol event (not a multi-event!) has been set multiple times.
Red	marks	">> M:"	zzz
White	events	">> E:" or "∗ E:"	zzz
White	marks	">> M:"	zzz

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, 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