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. Files in '.DLP' formate are provided by Orobors, '.DLPU' files are user-modified versions (see A: Export DL-Protocol and 'Edit DL-Protocol' window) of a specific '.DLP' file. When a protocol is initially selected, the button ist active and enables to automatically open the protocol file in another DatLab instance to see a protocol specific example data plot with properly set marks and events. Clicking the button removes the selected 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 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.
- 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.
- The button opens the 'Edit DL-Protocol' window (see below) to make changes in events and marks in the DL-Protocol.
- Below the buttons, the 'name field' automatically displays the name of the loaded protocol.
- 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.
- 'Edit DL-Protocol' window. Changes cna be made in concentration and titration volume of the events and marks in the specific DL-Protocol. 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. The active plot is indicated in the according name field (window, top). A second name field displays the DL-Protocol name, furthermore, a hyperlink (in blue) to the biobast is avialable. Similar to the DL-Protocol window, mark names are preceded by ">> M:", events are indicated by ">> E:" or "* E:" (multi-events).
- These changes can be saved to generate a modified DL-Protocol, see A: Export DL-Protocol.
- Below the buttons, the 'name field' automatically displays the name of the loaded protocol.
Running a DL-Protocol
- DL-Protocol will inform you if a titration event is missed or a repetition of a non multiple titration occured (highlighted in red).
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