Agilent MitoXpress Xtra Quick Start Guide
MitoXpress Xtra
Quick Start Guide
Five Step Workflow
Table of Contents
1. Plate Reader Setup 3
Find your plate reader download packet 3
Import protocol/template 3
Define the detection mode 3
2. Signal Optimization 4
Plate reader signal optimization 4
Plate preparation 4
Calculations 5
Adjustments for improved signal detection (S:B and signallevel) 6
Confirmation 6
3. Sample Optimization 7
Adherent cell titration plate 7
Suspension cell titration plate 8
Calculations 9
Adjustments for improved O2 consumption (OCR) 10
4. Running the Assay 11
Example control compound treatment (and FCCP optimization) 11
Day before measurement cell plate preparation 12
Control compound preparation 12
Assay plate preparation 12
5. Data Analysis 13
Guideline for applying data analysis tools to raw data 13
Data analysis tool options 13
This Quick Start provides all you need to know from plate
reader setup and signal optimization, to sample and compound
optimizations, as well as routine assay runs and data analysis.
For detailed instructions on product use, please see the
MitoXpress Xtra user manual.
1
Plate reader
setup
2
Signal
optimization
3
Sample
optimization
4
Running
the assay
5
Data
analytics
2
1. Plate Reader Setup
Key steps
1.1 Find your plate reader
download packet
1.2 Import protocol/template
1.3 Define the detectionmode
Step 1.1
Find your plate reader download packet
Find your Plate Reader online.
The download packet found in the link above contains the required collateral
and software templates you will need, including the recommended protocol
template files. These files contain default settings for your fluorescence plate
reader model, where available, or an instrument setup guide where not available.
Separately, it will contain a data analysis template specific to your plate reader
control software or, where that is not feasible, provide the data visualization tool
(Excel Macro). In addition, the MitoXpress Xtra user guide is provided, containing
detailed assay instructions.
Step 1.2
Import protocol/template
Import protocol templates into your plate reader software for easy setup. Open
the protocol and define the filter locations specific to your instrument filter
wheel/slide/cube and get started.
If a template is not provided, create a protocol in the instrument software,
inputting the instrument parameters described in the specific instrument
setupguide.
Ensure that software versions are up to date and compatible before starting.
Step 1.3
Define the detection mode
Decide/define which detection mode you intend to employ depending on the plate
reader specification used (basic, standard TR-F, and dual-read TR-F). See the user
guide contained in the download packet to inform your choice.
Use the recommended detection mode for your chosen plate reader
specifications, while ensuring that the correct excitation and emission filters are
installed correctly.
In cases where dual-read TR-F detection is recommended but the filters are not
available, it is possible to employ the standard TR-F detection as an alternative.
This is done using the monochromator rather than filters for excitation and
emission wavelength selection.
3
2. Signal Optimization
MX pr
Blank:
Key steps
2.1 Read; 20 minutes at 37°C
2.2 Calculations
i. S:B ratio (>3:1)
ii. Signal level (10 to
20% max. instrument
intensity (RFU))
iii. Lifetime signal level
(22to 26 µs)
2.3 Adjustments
2.4 Confirmation (repeatread)
Step 2.1
Plate reader signal optimization
Measure a cell-free signal optimization plate for verification of the protocol
template/instrument settings implemented in Step 1. This can be done with a
quick assessment of the signal-to-blank (S:B) and signal level (RFU).
• The recommended S:B is >3:1
• The signal level must be ~10 to 20% maximum intensity (RFU) on the plate
reader (~15% of saturation). Otherwise, the signal will overflow/saturate the
instrument.
Usually the default protocol parameters will provide a suitable S:B and RFU;
however, in the unlikely event it is outside these recommendations, simple
adjustments can be made, found in Step 2.3.
Step 2.1.1
Plate preparation
Prepare and read a signal optimization plate using the plate layout in Figure 1.
MitoXpress probe, media, and HS mineral oil preparation are described in the
MitoXpress Xtra user guide.
12345678910 11 12
MX
MX
MX
A
probe
probe
probe
BlankBlank BlankBlank
B
C
D
E
F
G
H
obe: 10 µL MitoXpress probe + 90 µL Media + 100 µL HS mineral oil
100 µL Media + 100 µL HS mineral oil
Figure 1. Signal optimization plate layout.
MX
probe
Note: It is important that the media/sample, HS mineral oil, microplate, and plate
reader are prepared and maintained at the desired assay temperature, 37 °C. The
HS mineral oil should be warmed to 37 °C before the assay for easier use.
4
Tip: For consistent, reliable, and quick HS mineral oil dispensing, a repeater
Signal (RFU)
Signal
Blank
type pipette is recommended (using a 1.25 mL syringe tip or 2 mL combitip).
Prepare the repeater syringe tip by trimming ~3 to 4 mm off the tip at a 45°
angle. Remove the internal nozzle from the oil dropper bottle and slowly pick up
the prewarmed HS mineral oil (avoid pipetting up and down, as this can cause
bubbles) and dispense 100 μL into each well at an angle of ~45°, allowing the oil
to flow down the side of each well.
Pipetting tips are found on page 36 of the MitoXpress Xtra user guide.
Key steps
2.1 Read; 20 minutes at 37°C
2.2 Calculations
i. S:B ratio (>3:1)
ii. Signal level (10 to
20% max. instrument
intensity (RFU))
iii. Lifetime signal level
(22to 26 µs)
2.3 Adjustments
2.4 Confirmation (repeatread)
MitoXpress signal
RFU
Step 2.2
Calculations
Perform calculations i. to iii. using one of the following data analysis options:
(a) Plate reader software analysis/templates
(b) Agilent Data Visualization Tool
Review the raw results and calculate the following:
i. Signal:blank ratio: Using average RFU values measured at the 20-minute
time point, calculate signal RFU (row A)/blank RFU (row B). Ensure
that the S:B ratio is >3. If using a TRF detection mode: S:B >5 to 10 is
achievable.
ii. RFU signal level: Using RFU signal at the 20-minute time point, the
measured RFU signal level for the MX probe samples should be in the
range of ~10 to 20% of the maximum instrument intensity (RFU). See
the plate reader user manual or software help section to identify the
arbitrary maximum/saturated RFU signal level for the given plate reader
model.
iii. Lifetime signal (µs) level: MitoXpress assay signal (21% O2) ~22 to
26µs.
Blank
Note: This calculation is only applicable when using the advanced dual-read TRF
detection mode on a filter-based plate reader.
Time
3000
Decision point:
• If the recommended S:B ratio, signal level, and lifetime values (where
applicable) are successfully achieved, we recommended proceeding to Step 3.
2500
1000
1500
S:B = 11
1000
500
0
• If, however, the recommended S:B ratio, signal level, or lifetime values are not
achieved, we suggest proceeding to Step 2.3 Adjustments.
5
Loading...