Bio-Rad Bio-Plex® Assay Builder User Manual
Page 10
Section 7
Data Acquisition
Recommendations for acquiring data using the Bio-Plex suspension array
system are listed below. Alternatively, refer to the Bio-Plex Manager
software user guide or the instructions provided with the Luminex 100
instrument.
Prepare System
1.
Empty the waste bottle and fill the sheath fluid bottle before starting.
This will prevent fluidic system backup and potential data loss.
2.
Turn on the reader and microplate platform (and HTF if present).
Allow the system to warm up for 30 min.
3.
Select Startup and follow the instructions to prepare the reader
to acquire data. If the system is idle for 4 hr the lasers will
automatically turn off and a 30 min warm-up period will again be
required prior to acquiring data. Select Warm up and wait for
the optics to reach operational temperature.
Calibrate With High RP1 Target Value
Calibrate using Bio-Plex calibration beads and target values. Daily
calibration is recommended before acquiring data.
1.
Select Calibrate and confirm that the default values for CAL1
and CAL2 are the same as the values on the Bio-Plex calibration
bead labels. Use the Bio-Plex High RP1 target value for CAL2
calibration for Bio-Plex phosphoprotein and total target assays.
NOTE: When acquiring data for Bio-Plex phosphoprotein or total
target assays with a Luminex 100, Luminex Data Collector software,
and Luminex calibration beads, it is necessary to convert the Luminex
CAL2 calibration bead RP1 target value using the following equation:
Bio-Plex High RP1 target value = (Luminex RP1 target value) x 4.55
Add the new target value to the Luminex software by selecting
Calibrate, then New under the Reporter Channel in the Start
Calibration dialog. Enter the new target value and save it as a new
lot. Then calibrate using the new RP1 target value.
15
5.
The next day, prepare a sufficient volume of detection antibodies (1x)
using
detection antibody diluent
(see the example on the previous
page). Each well requires 1µl of detection antibodies (25x) for each
target adjusted to a final volume of 25 µl (refer to the table on the
previous page).
6. After the incubation, slowly remove and discard the sealing tape,
then vacuum-filter.
7. Wash 3 times.
8. Vortex the detection antibodies gently and add 25 µl to each well,
changing the pipet tip after every volume transfer. Incubate for 30 min.
9.
After the incubation, slowly remove and discard the sealing tape,
then vacuum-filter.
10. Wash 3 times.
11. Keep the plate in the dark and prepare a sufficient volume of
streptavidin-PE (1x) using
wash buffer
(see example below). Each
well requires 0.5 µl of streptavidin-PE (100x) adjusted to a final
volume of 50 µl. Store in the dark after preparation.
12. Vortex the diluted streptavidin-PE vigorously and add 50 µl to each
well. Incubate for 10 min.
13. After the incubation, slowly remove and discard the sealing tape,
then vacuum-filter.
14. Rinse 3 times.
15. Add 125 µl of
resuspension buffer
to each well. Incubate for 30 sec.
If the data is not acquired immediately, the assay may be stored in
the dark at 4ºC for up to 24 hrs.
14
Example Streptavidin-PE Calculations
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