Run DL-Protocol/Set O2 limit: Difference between revisions
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{{MitoPedia | {{MitoPedia | ||
|abbr= | |abbr=DLP | ||
|description='''DL-Protocols''' can be selected in DatLab | |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 - DatLab |Events]] and [[Marks - DatLab |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 titration]]s (see [[MitoPedia: SUIT]]) proceed stepwise to activate a sequence of [[coupling control state]]s and [[pathway control state]]s. 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 code - DatLab |User]] logged into DatLab. A '''Lower O2 limit [ยตM]''' 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. | ||
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{{MitoPedia O2k and high-resolution respirometry | {{MitoPedia O2k and high-resolution respirometry | ||
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== Protocol menu == | == Protocol menu == | ||
[[File:Protocol-menu.jpg|thumb|right|200px|'''Protocol menu''']] | [[File:Protocol-menu.jpg|thumb|right|200px|'''Protocol menu''']] | ||
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:::: 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. | :::: 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[ยตM]' | :::: '''Lower O2 limit [ยตM]''': 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. | ||
[[File:Warning-O2.png|thumb|right|200px|'''O2-limit warning button''']] | [[File:Warning-O2.png|thumb|right|200px|'''O2-limit warning button''']] | ||
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Revision as of 11:41, 14 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 [ยตM] 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-Procotol 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 DL-protocols (.DLP or DLPU) can be selected () for chamber A and B. 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 [ยตM]: 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.
- 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, as it is necessary for titrating the uncoupler (U) in SUIT protocols.
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 [ยตM] as specified.
- Titration volume, v [ยตl]
- Chamber volume, V [ยตl]
- Final concentration, cB [mM] or [ยตM] as specified.
- cB [mM] = cB,in [mM] x v [ยตl] / V [ยตl]
- Example: Malate (M), cM,in = 800 mM stock; v = 5 ยตl titration volume; V = 2,000 ยตl chamber volume.
- cM [mM] = cM,in [mM] x v [ยตl] / V [ยตl] = 800 mM x 5 ยตl / 2,000 ยตl = 2 mM