10010584

10010584
Bio-Plex
Manager™ software
recommended
Bio-Plex Pro Assays
Angiogenesis
TM
Instruction Manual
For technical support, contact your local Bio-Rad office or
in the US, call 1-800-4BIORAD (1-800-424-6723).
For research use only. Not for diagnostic procedures.
Table of Contents
Section 1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Section 2
Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Section 3
Product Description . . . . . . . . . . . . . . . . . . . . . . .4
Section 4
Required Materials . . . . . . . . . . . . . . . . . . . . . . . . 5
Section 5
Recommended Materials . . . . . . . . . . . . . . . . . . 6
Section 6
Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . 7
Section 7
Standard Preparation . . . . . . . . . . . . . . . . . . . . . 8
Section 8
Control Preparation (optional) . . . . . . . . . . . . . 10
Section 9
Assay Instructions . . . . . . . . . . . . . . . . . . . . . . .
Plan Experiment . . . . . . . . . . . . . . . . . . . . . . . . . .
Prepare Coupled Magnetic Beads . . . . . . . . . . . .
Calibrate Vacuum Apparatus . . . . . . . . . . . . . . . .
Assay Procedure . . . . . . . . . . . . . . . . . . . . . . . . . .
Section 10
Data Acquisition . . . . . . . . . . . . . . . . . . . . . . . . . 17
Section 11
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . 24
Section 12
Safety Considerations . . . . . . . . . . . . . . . . . . . .28
12
12
13
13
14
Section 1
Introduction
Angiogenesis is a critical component of various normal and pathological
processes. A regulated angiogenic process includes the growth of new
blood vessels for tissue repair, fetal development and female reproductive
cycle. An uncontrolled angiogenic growth contributes to a major
pathological component of diseases such as cancer, cardiovascular
disease, and rheumatoid arthritis. Many proteins involved in the
uncontrolled angiogenic growth are candidate drug targets relevant for the
development of cancer therapies. Developing these therapies involves
measurement of various angiogenesis biomarkers.
Bio-Plex Pro™ angiogenesis assays are magnetic bead-based multiplex
assays that allow the measurement of angiogenesis biomarkers in diverse
matrices including serum, plasma, and cell/tissue culture supernatant. The
multiplexing feature makes it possible to quantitate the level of multiple
angiogenesis targets in a single well of a 96-well microplate in just 3 hours,
using as little as 12.5 µl of serum or plasma, or 50 µl of culture
supernatant.
As one of the most recent additions to the Bio-Plex® suspension array
system, these assays incorporate magnetic beads into their design. The
magnetic beads allow the use of an assay protocol similar to non-magnetic
Bio-Plex assays, with the option of using magnetic separation of wash
steps instead of vacuum filtration (and allows automation of many of the
steps).
Bio-Plex Manager™ software is recommended for Bio-Plex Pro
angiogenesis assays. Instructions for Luminex xPONENT software are
also provided. For instructions using other xMAP system software
packages, contact Bio-Rad Technical support or your Bio-Rad field
application specialist.
For a current listing of Bio-Plex angiogenesis assays, visit us on the Web at
www.bio-rad.com/bio-plex/
1
Section 2
Principle
Technology
The Bio-Plex® suspension array system is built around three core
technologies. The first is a novel technology that uses up to 100 unique
fluorescently dyed beads (xMAP technology) that permit the simultaneous
detection of up to 100 different types of molecules in a single well of a
96-well microplate. The second is a flow cytometer with two lasers and
associated optics to measure the different molecules bound to the
surface of the beads. The third is a high-speed digital signal processor
that efficiently manages the fluorescent output.
Assay Format
The principle of these 96-well plate-formatted, bead-based assays is
similar to a capture sandwich immunoassay. An antibody directed
against the desired angiogenesis target is covalently coupled to internally
dyed beads. The coupled beads are allowed to react with a sample
containing the angiogenesis target. After a series of washes to remove
unbound protein, a biotinylated detection antibody specific for a different
epitope is added to the reaction. The result is the formation of a
sandwich of antibodies around the angiogenesis target. Streptavidinphycoerythrin (streptavidin-PE) is then added to bind to the biotinylated
detection antibodies on the bead surface.
Data Acquisition and Analysis
Data from the reaction are then acquired using the Bio-Plex suspension
array system (or Luminex system), a dual-laser, flow-based microplate
reader system. The contents of the well are drawn up into the reader.
The lasers and associated optics detect the internal fluorescence of the
individual dyed beads as well as the fluorescent signal on the bead
surface. This identifies each assay and reports the level of target protein
in the well. Intensity of fluorescence detected on the beads indicates the
relative quantity of targeted molecules. A high-speed digital processor
efficiently manages the data output, which is further analyzed and
presented as fluorescence intensity on Bio-Plex Manager™ software.
2
Assay Workflow
Prewet wells
Add beads
Wash
Add standards, controls,
and samples, 30 min
Wash
Add detection antibody, 30 min
Wash
Add streptavidin-PE, 10 min
Wash
Resuspend, acquire data
3
Section 3
Bio-Plex® Reagent Kit
Product Description
Bio-Plex Pro™ angiogenesis assays require the use of the Bio-Plex®
angiogenesis reagent kit to run the multiplex panel.
Component of the
Bio-Plex Angiogenesis
Reagent Kit
171-304060
1x96-Well Format
171-304061
10x96-Well Format
Bio-Plex assay buffer
Store at 4ºC. Do not freeze.
1 x 75 ml
1 x 750 ml
Bio-Plex wash buffer
Store at 4ºC. Do not freeze.
1 x 150 ml
2 x 750 ml
Bio-Plex detection
antibody diluent
Store at 4ºC. Do not freeze.
1 x 15 ml
1 x 150 ml
Streptavidin-PE (100x)
Store at 4ºC. Do not freeze.
1 vial
1 vial
1 plate
10 plates
1 pack of 4
10 packs of 4 (40)
1
1
Sterile filter plate (96-well)
with cover and tray
Sealing tape
Angiogenesis Instruction
Manual
Storage and Stability
Kit components should be stored at 4ºC and never frozen. Coupled
magnetic beads and streptavidin-PE should be stored in the dark. All
components are guaranteed for up to 6 months from the date of
purchase when stored as specified in this manual.
4
Section 4
Required Materials
Bio-Plex Pro™ angiogenesis assays require the Bio-Plex Pro™
angiogenesis reagent kit and a multiplex angiogenesis assay panel. The
Bio-Plex Pro angiogenesis assay panels contain the following
components.
•
Antibody-conjugated beads (25x concentration)
•
Detection antibody (10x concentration)
•
Angiogenesis standard (2 vials, lyophilized)
•
Angiogenesis control (2 vials, lyophilized)
Visit us on the web at www.bio-rad.com/bio-plex/ for our latest list of
assays.
5
Section 5
Recommended Materials
Item
Bio-Plex human serum diluent kit
For tissue culture samples, dilute
samples and standards with appropriate
tissue culture media
Bio-Plex Pro human angiogenesis
standards
Additional standards sold separately
(optional)
Item
Bio-Rad catalog #171-305000
(1x96)
Bio-Rad catalog #171-305001
(10x96)
Bio-Rad catalog #171-D40003
(2 vials, lyophilized)
Bio-Rad catalog #171-D40004
(50 vials,
lyophilized)
Ordering
Information
Bio-Plex® suspension array system
(or Luminex System)
Bio-Rad catalog #171-000205
Bio-Plex validation kit
Bio-Plex calibration kit
Bio-Rad catalog #171-203001
Bio-Rad catalog #171-203060
Microtiter plate shaker
IKA-Schuttler MTS-4 shaker for 4
microplates or Lab-Line Model 4625
Plate Shaker (or equivalent, capable of
300-1,100 rpm)
Filter plate vacuum apparatus
Bio-Rad Aurum™ vacuum manifold
IMPORTANT: The use of filter plate
manifolds other than the one specified
may result in diminished assay
performance; see section 8 for
instructions specific to this assay
Vortexer
VWR brand mini-vortexer
Scientific Instruments Vortex-Genie 2 mixer
Reagent reservoir
Corning, Inc. Costar 50 ml reagent
reservoir 4870
Other materials
Pipets and pipet tips, sterile distilled
water, aluminum foil, absorbent paper
towels, 1.5 ml microcentrifuge tubes,
15 ml culture tubes
6
Ordering Information
IKA catalog #3208000
VWR catalog #57019-600
Bio-Rad catalog #732-6470
VWR catalog #58816-121
VWR catalog #58815-234
Bio-Rad catalog #224-4872
Section 6
Sample Preparation
This section provides instructions for preparing samples derived from serum,
plasma, and tissue culture supernatant. For sample preparations not
mentioned here, consult the publications listed in Bio-Rad bulletin 5297,
available for download at discover.bio-rad.com
Serum and Plasma Samples
Note that for plasma samples, both EDTA plasma and citrate plasma are
acceptable. Avoid using hemolyzed samples.
1.
Collect and process the serum or plasma samples and assay immediately
or freeze at –20ºC. Avoid repeated freeze-thaw cycles.
2.
Centrifuge the samples at 13,200 rpm for 10 min at 4ºC to clear the
samples of precipitate. Alternatively, carefully filter the samples with a
0.8/0.22 µm dual filter to prevent instrument clogging.
3.
Immediately dilute 1 volume of sample with 3 volumes of sample diluent.
Keep the samples on ice until ready for use.
Tissue Culture Supernatant Samples
1. Collect and process the tissue culture supernatant samples and assay
immediately or freeze at –20ºC. Avoid repeated freeze thaw cycles.
2.
If required, dilute the culture supernatant with culture medium.
Serum-free culture medium should contain carrier protein (such as BSA)
at a concentration of at least 0.5%. Keep the samples on ice until ready
for use.
7
Section 7
Standard Preparation
Two vials of Iyophilized angiogenesis standards are provided in each Bio-Plex
Pro™ angiogenesis assay. However, only one vial is required per 96-well plate.
The product insert provided with the assay lists the concentration of the
reconstituted standard. This procedure will prepare enough standard to run
each dilution in duplicate.
Reconstitute Standards
1. Gently tap the glass vial containing the lyophilized angiogenesis
standard on a solid surface to ensure the pellet is at the bottom.
2.
Reconstitute 1 vial of lyophilized standard with 500 µl of the
standard diluent. Do not use assay buffer to dilute standards.
3.
Gently vortex 1–3 sec and incubate on ice for 30 min.
4.
Be consistent with the incubation time for optimal assay performance.
Prepare Standard Dilution Series
The angiogenesis concentrations specified for the 8-point standard
dilution set have been selected for optimized curve fitting using the 5parameter logistic (5PL) or 4-parameter logistic (4PL) regression in
Bio-Plex Manager™ software. Results generated using dilution points
other than those listed in this manual have not been optimized.
1.
Label a set of 1.5 ml Eppendorf tubes as shown in the diagram on
the next page.
2.
Pipet the appropriate volume of standard diluent into the tubes (see
diagram on next page). Use serum standard diluent for serum and
plasma samples and culture medium for culture samples.
8
3.
Add 400 µl of reconstituted standard to the first 1.5 ml tube with
0 µl of standard diluent. This is identified as S1 in the diagram below
and in the product insert provided with assay.
4.
Continue making serial dilutions of the standard as shown. After
making each dilution, vortex gently and change the pipet tip after every
transfer.
NOTE: Running an additional two 0 pg/ml blanks is strongly
recommended. The blank wells are useful for troubleshooting and
determining LOD. Use 50 µl of the appropriate standard diluent as
the blank sample. The 0 pg/ml points should be formatted as
blanks, not as points in the curve, when using Bio-Plex Manager
software. Formatting these wells as blanks automatically subtracts
the background MFI values from the reading, and may result in
negative MFI values in some wells. If negative MFI values are
undesirable, format the 0 pg/ml wells as background controls.
5.
Keep the standards on ice until ready for use. Standards should be
used immediately and should not be frozen for future use.
Standard Dilution Series
9
Section 8
Control Preparation (Optional)
Two vials of lyophilized angiogenesis control are provided in each Bio-Plex
Pro™ Angiogenesis assay. The preparation of high, medium, and low controls
is optional to monitor plate-to-plate variations. This section provides
instructions on how to reconstitute the Iyophilized controls (refer to the product
insert provided with the assay for reconstituted control values). The
reconstituted control can then be further diluted to prepare any concentration of
user-specified quality controls.
Reconstitute Angiogenesis Controls
To ensure optimal assay performance, angiogenesis controls should be
prepared in a manner consistent as that used to prepare the angiogenesis
standards.
1.
Gently tap the glass vial containing the lyophilized angiogenesis
control on a solid surface to ensure the pellet is at the bottom.
2.
Reconstitute 1 vial of lyophilized control with 800 µl of the appropriate
diluent. Do not use assay buffer to dilute controls. This is identified
as stock in the product insert provided with the assay.
3.
Gently vortex 1–3 sec and incubate on ice for 30 min. Be consistent
with the incubation time to ensure optimal assay performance.
4.
The reconstituted angiogenesis control should be further diluted to
create the desired QC samples in the same diluents specified in the
table below. Concentration of the reconstituted angiogenesis control
can be found in the product insert provided with the assay.
10
Example of Control Dilution Series
11
Section 9
Assay Instructions
The following instructions apply to Bio-Plex Pro™ angiogenesis assays. All
of the necessary components are provided premixed for ease of use.
Plan Experiment
1. Assign which wells of a 96-well plate will be used for each standard,
control, and sample (see the example below).
2.
Determine the total number of wells that will be used in the assay.
Include a 25% excess (or add 2 wells for every 8 wells used) to
ensure that enough diluted coupled beads, detection antibodies,
and streptavidin-PE are prepared.
Example Plate
12
Prepare Coupled Magnetic Beads
Protect the beads from light by covering the tubes with aluminum foil.
Keep all tubes on ice until ready to use.
1.
Vortex the coupled beads (25x) at medium speed for 15-20 sec.
2.
Prepare a sufficient volume of coupled beads (1x) using assay
buffer. Each well requires 2 µl of coupled beads (25x) adjusted to a
final volume of 50 µl with assay buffer (refer to the example below).
Example Bead Calculations
# of Wells
25x Beads (µl)
Assay Buffer (µl)
Total Volume (µl)
96
240
5,760
6,000
48
120
2,880
3,000
32
80
1,920
2,000
24
60
1,440
1,500
Calibrate Vacuum Apparatus
The vacuum apparatus must be calibrated at the beginning of the assay
to ensure an optimal bead yield. For more detailed instructions, refer to
the Bio-Plex® suspension array system hardware instruction manual.
1.
Prewet all the wells of a 96-well filter plate with 100 µl of assay buffer.
2.
Place the filter plate on the vacuum apparatus and turn on the
vacuum to the maximum level.
3.
Press on the filter plate and note the time required to remove the
buffer from the wells by vacuum filtration. The evacuation time
should be 2–5 sec.
If the evacuation time is <2 sec, the pressure is too high. Open the
vacuum control valve slightly and repeat steps 1–3.
If the evacuation time is >5 sec, the pressure is too low. Close the
vacuum control valve slightly and repeat steps 1–3.
13
Assay Procedure
Bring all buffers to room temperature. Avoid bubbles when pipetting.
Assay Key – The following terms are repeated throughout the assay
procedure. Refer to these detailed instructions when wash, incubate, and
vacuum-filter are shown in bold.
Add 100µL of wash buffer to each well. Place the filter plate on a calibrated vacuum
apparatus and remove the buffer by vacuum filtration. Blot the bottom of the filter plate
with a clean paper towel. Repeat as specified.
Tip: Thoroughly blot the bottom of the filter plate with a clean paper towel to
prevent cross-contamination and plate leakage.
Gently cover the filter plate with a new sheet of sealing tape. Place the filter plate on a
microplate shaker and then cover with aluminum foil. Shake the filter plate at room
temperature at 1,100 rpm for 30 sec, then at 300 rpm for the specified incubation time.
Tip: Apply sealing tape gently on the filter plate (e.g. press down only on edges) to
prevent positive pressure inside the wells that could lead to plate leaking during shaking.
To avoid splashing of samples that may lead to cross-well contamination, slowly ramp
up the shaker to the maximum speed before incubation. Cover plate with aluminum foil
to prevent photobleaching.
Place the filter plate on a calibrated vacuum apparatus and remove the buffer by
vacuum filtration. Blot the bottom of the filter plate with a clean paper towel.
Tip: Visually examine each well to ensure that buffers are completely drained from each well.
1.
Equilibrate the diluted standards, samples, and controls at room
temperature for 20 min prior to use.
2.
Prewet and block the desired number of wells in a 96-well filter plate
with 100 µl of assay buffer and vacuum-filter. If fewer than 96
wells are required, mark the plate to identify the unused wells for
later use and cover the unused wells with sealing tape.
3.
Vortex the coupled magnetic beads (1x) for 15–20 sec at medium
speed. Add 50 µl to each well and immediately vacuum-filter.
4.
Wash twice.
5.
Gently vortex the diluted standards, controls, and samples for 1–3 sec.
Add 50 µl of standard, control, or sample to each well, changing the
pipet tip after every volume transfer. Incubate for 30 min.
6.
While the samples are incubating, perform a 30 sec quick-spin
centrifugation of the detection antibody (10x) prior to pipetting to
collect the entire volume at the bottom of the vial.
14
7.
Prepare a sufficient volume of detection antibodies (1x) using
detection antibody diluent. Each well requires 2.5 µl of detection
antibodies (10x) adjusted to a final volume of 25 µl with detection
antibody diluent (refer to the example below).
Example Detection Antibody Calculations
# of Wells
10x Detection
Antibody (µl)
Detection Antibody
Diluent (µl)
Total Volume (µl)
96
300
2,700
3,000
48
150
1,350
1,500
32
100
900
1,000
24
75
675
750
8.
After incubating the samples, slowly remove and discard the sealing
tape, then vacuum-filter.
9.
Wash 3 times.
10. Vortex the detection antibodies gently and add 25 µl to each well.
Incubate for 30 min.
11. While the detection antibodies are incubating, perform a 30 sec
quick-spin centrifugation of the streptavidin-PE (100x) prior to
pipetting to collect the entire volume at the bottom of the vial.
12. Prepare a sufficient volume of streptavidin-PE (1x) using assay buffer.
Each well requires 0.5 µl of streptavidin-PE (100x) adjusted to a final
volume of 50 µl with assay buffer (refer to the example on the
following page).
15
Example Streptavidin-PE Calculations
100x
Assay Buffer (µl)
Streptavidin-PE
# of Wells
(µl)
Total Volume (µl)
96
60
5,940
6,000
48
30
2,970
3,000
32
20
1,980
2,000
24
15
1,485
1,500
13. After the detection antibody incubation, slowly remove and discard
the sealing tape, then vacuum-filter.
14. Wash 3 times.
15. Vortex the streptavidin-PE (1x) vigorously and add 50 µl to each well.
Incubate for 10 min.
16. After the streptavidin-PE incubation, slowly remove and discard the
sealing tape, then vacuum-filter.
17. Wash 3 times.
18. Add 125 µl of assay buffer to each well. Cover the filter plate with a
new sheet of sealing tape. Shake the filter plate at room temperature
at 1,100 rpm for 30 sec and remove the sealing tape before
reading the plate.
16
Section 10
Data Acquisition
Bio-Plex Manager™ software is recommended for Bio-Plex Pro™
angiogenesis assays. Refer to the details in the appropriate section below
for your xMAP system software package. For instructions using other
xMAP system software packages, contact Bio-Rad technical support or
your Bio-Rad field applications specialist.
NOTE: To minimize protocol setup, lot-specific Bio-Plex Pro
angiogenesis assay protocols for Bio-Plex Manager version 4.0 and higher
are available for download at www.biorad.com/bio-plex/standards.
Prepare System
1. Empty the waste bottle and fill the sheath fluid bottle before starting
(if HTF not present). This will prevent fluidic system backup and
potential data loss.
2.
Turn on the reader, XY platform, and HTF (if included). Allow the
system to warm up for 30 min.
3.
Select Start up
and follow the instructions to prepare the reader
to acquire data. If the system is idle for 4 hr without acquiring data, the
lasers will automatically turn off. To reset the 4-hour countdown, select
Warm up
and wait for the optics to reach operational
temperature.
Calibrate With Low RP1 Target Value
Calibrate using Bio-Plex calibration beads and target values. Bio-Plex Pro
angiogenesis assays are run using the low RP1 target value on the Cal 2
bottle. Refer to the details in the appropriate section below for your xMAP
system software package.
Bio-Plex Manager Software version 5.0 and higher
These software versions require calibration only with the low RP1
target value. Daily calibration is recommended before acquiring data.
17
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 low RP1
target value.
2.
Select OK and follow the instructions for CAL1 and CAL2
calibration.
Bio-Plex Manager Software version 4.0, 4.1 and 4.1.1
This software versions permit calibration at either low or high
RP1 target values. The selection of which target value to use is
assay dependent. For Bio-Plex Pro angiogenesis assays,
calibrate using the low RP1 target value on the Cal 2 bottle. Recalibration is necessary between assays requiring different RP1
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 BioPlex calibration bead labels. Use the Bio-Plex low RP1
target value.
2.
Select OK and follow the instructions for CAL1 and CAL2
calibration.
Prepare Protocol
Lot-specific assay protocols are available for download at www.biorad.com/bio-plex/standards. If the desired protocol is not available for
download, create a new protocol.
1.
Open a new protocol by selecting File, then New from the main
menu. Locate the steps at the left of the protocol menu.
2.
Select Step 1 (Describe Protocol) and enter information about the
assay.
3.
Select Step 2 (Select Analytes) and choose the appropriate
angiogenesis panel name, if available in the menu. If the panel name
is not available, create a new panel:
18
a)
Click the Add Panel button
in the Select Analytes toolbar.
b)
Enter a name for the new panel in the top field. Select the “BioPlex Pro” assay type from the pull down menu (only for version
5.0 or higher). Then click Add to add analytes.
c)
Enter the bead region number of the first analyte in the Region
field, and the analyte name in the Name field.
NOTE: The bead region number must be correct for proper
detection of analytes. Using the product insert provided with the
assay, confirm that this number is correct before proceeding.
d)
Click Add Continue to add the analyte to the panel and continue
adding more analytes. When you have entered your last analyte,
click the Add button to add it to the list.
e)
When you are finished creating the panel, click OK to save your
changes and return to the Select Analytes window.
4.
After selecting the panel, move analytes to the Selected view by
choosing from analytes in the Available view using the Add button or
Add All button. Note that the analytes will already be entered in the
Selected view when using a downloaded protocol.
5.
Select Step 3 (Format Plate) and click on the Plate Formatting tab.
Select the number of replicates and the auto fill direction. Then click
the
icon and drag the cursor over all the wells that contain
standards. Then click on the
icon and drag the cursor over the
wells that contain blanks. Repeat with
and
to identify all the
wells that contain controls and samples.
NOTE: If the protocol was downloaded from the website, the
standards will already be added to the plate format. Make any
necessary changes to the plate layout to match your plate
requirements.
19
Plate Formatting Example
6.
20
Select Step 4 (Enter Standards Info) to enter standards information.
Skip to the next step if you are using a downloaded protocol from
the website; this information will already be entered.
a)
Deselect the box labeled “same concentration values for all
analytes”.
b)
Select the first analyte from the pull-down cell.
c)
Click on the Enter Automatically button and then select the most
concentrated value (typically S1).
d)
Enter the value for the highest concentration. The information is
included on the product insert provided with the assay.
e)
Enter the dilution factor (usually 4) and select Calculate. The
concentrations for each replicate point of the standard will be
populated for the selected analyte.
f)
Repeat steps 6b through 6e for each analyte in the assay.
7.
Select Step 5 (Enter Controls Info) to enter controls information. This
is where the concentration of the user-specified controls is entered
into the protocol.
a)
If necessary, deselect the box for same concentration values for
all analytes.
b)
Select an analyte from the pull down cell.
c)
Enter the description, concentration, and dilution information for
each user-specified control.
d)
Repeat steps 7b and 7c for each additional analyte in the assay.
8.
Select Step 6 (Enter Sample Info) and enter sample information.
Remember to enter information for the appropriate dilution factor.
9.
Select Step 7 (Run Protocol).
Acquire Data
1. Shake the assay plate at 1,100 rpm for 30 sec immediately before
acquiring data.
2.
Visually inspect the plate and ensure that the assay wells are filled
with buffer prior to placing the plate in the Bio-Plex microplate platform.
3.
Slowly remove the sealing tape and any plate cover before placing
the plate in the reader.
4.
Select Step 7 (Run Protocol) and refer to the details in the
appropriate section below for your xMAP system software package:
Bio-Plex Manager Software version 4.1, 4.1.1 and 5.0
a) Specify data acquisition for 100 beads per region.
b)
In Advanced Settings, confirm that the Bead Map is set to
25 region.
c)
In Advanced Settings, set the sample size to 50 µl.
21
d)
In Advanced Settings, confirm that the default DD gate
values are set to 5000 (low) and 32000 (high).
e)
Select Start, save the .rbx file, and begin data acquisition.
Bio-Plex Manager Software version 4.0
Note that this version does not include the 25 Bead Map. Hence,
the 100 bead map is used.
5.
a)
Specify data acquisition for 100 beads per region.
b)
In Advanced Settings, set the sample size to 50 µl.
c)
In Advanced Settings, confirm that the default DD gate
values are set to 5000 (low) and 32000 (high).
d)
Select Start, save the .rbx file, and begin data acquisition.
If acquiring data from more than one plate, empty the waste bottle
and refill the sheath bottle after each plate (if HTF not present). Select
Wash Between Plates
and follow the instructions for fluidics
maintenance. Then repeat the Prepare Protocol and Acquire Data
steps.
NOTE: Use the Wash Between Plates command after every plate run
to reduce the possibility of clogging the instrument.
6.
22
When data acquisition is complete, select Shut Down
the instructions.
and follow
Reacquire Data
It is possible to aquire data from a well or plate a second time using the
Rerun/Recovery mode located below Start in Step 7 (Run Protocol).
1.
Check the wells where data will be acquired a second time. Any
previous data will be overwritten.
2.
Remove the buffer by vacuum filtration and add 100 µl of assay buffer
to each well. Cover the filter plate with a new sheet of sealing tape.
3.
Repeat Acquire Data steps 1-6 to acquire data a second time. The
data acquired should be similar to the data acquired initially;
however, the data acquisition time will be extended since fewer beads
are present in each well.
Luminex® xPONENT® Software
Luminex xPONENT software does not include the 25-bead map. Hence,
the 100-bead map is used. Refer to the detailed instructions in the
xPONENT software manual.
1.
Calibrate with xMAP MagPlex Classification Microspheres and xMAP
Reporter Calibration Microspheres (sold by Luminex).
2.
Verify with xMAP MagPlex Control Microspheres and xMAP Control
Microspheres (sold by Luminex).
3.
Create the angiogenesis panel protocol. Make sure to include the
correct bead region numbers and target names (refer to the product
insert).
3.
The 100-region map should be selected.
4.
Set the gate settings to 10,000 (low) to 22,500 (high).
23
Section 11
Troubleshooting Guides
This troubleshooting guide addresses problems that may be encountered with
Bio-Plex Pro™ angiogenesis assays. If you experience any of the problems listed
below, review the possible causes and solutions provided. This will assist you in
resolving problems directly related to how the assay steps should be performed.
Poor assay performance may also be due to the Bio-Plex® suspension array
reader. To eliminate this possibility, we highly recommend use of the Bio-Plex
validation kit. This kit will validate all the key functions of the array reader and assist
the user in determining whether or not the array reader is functioning properly.
Possible Causes
Possible Solutions
High Inter-Assay CV
Standards were not
reconstituted consistently
Incubate the reconstituted
standards for 30 min on ice. Always
be consistent with the incubation
time and temperature.
Reconstituted standards and
diluted samples were not stored
properly
Reconstituted standards and diluted
samples should be prepared on ice
as instructed. Prior to plating, the
reconstituted standards and diluted
samples should be equilibrated to
room temperature.
High Intra-Assay CV
Bottom of filter plate not dry
24
Dry the bottom of the filter plate with
absorbent paper towel (preferably
lint-free) to prevent crosscontamination.
Possible Causes
Possible Solutions
Pipetting technique
Pipet carefully and slowly when
adding standards, samples,
detection antibodies, and
streptavidin-PE, especially when
using a multichannel pipet. Use a
calibrated pipet. Change pipet tip
after every volume transfer.
Reagents and assay components
were not equilibrated to room
temperature prior to plating
All reagents and assay components
should be equilibrated to room
temperature prior to plating.
Contamination with wash
buffer during wash steps
During the wash steps, be careful
not to splash wash buffer from one
well to another. Be sure that the
wells are filtered completely and that
no residual volume remains. Also,
be sure that the microplate shaker
setting is not too high. Reduce the
microplate shaker speed to minimize
splashing.
Slow pipeting samples and
reagents across the plate
Sample pipeting across the entire
plate should take less than 4 min.
Reagent pipeting across the entire
plate should take less than 1 min.
25
Possible Causes
Possible Solutions
Low Bead Count
Miscalculation of bead dilution
Check your calculations and be
careful to add the correct volumes.
Beads clumped in multiplex
bead stock tube
Vortex for 15–20 sec at medium
speed before aliquoting beads.
Vacuum on for too long when
aspirating buffer from wells
Do not apply vacuum to the filter
plate for longer than 10 sec after the
buffer is completely drained from
each well.
Did not shake filter plate enough
before incubation steps and prior
to reading
Shake the filter plate at 1,100 rpm
for 30 sec before incubation steps
and immediately before reading
the plate.
Reader is clogged
Refer to the troubleshooting guide
in the Bio-Plex hardware
instruction manual.
Low Signal or Poor Sensitivity
Standards reconstituted incorrectly
Follow the standard preparation
instructions carefully (section 7).
Detection antibody or
streptavidin-PE diluted incorrectly
Check your calculations and be
careful to add the correct volumes.
26
Possible Causes
Possible Solutions
High Background Signal
Incorrect buffer was used
(for example, assay buffer
used to dilute standards)
Use sample matrix or serum
standard diluent to dilute
the standards.
Spiked “0 pg/ml” wells by mistake
Be careful when spiking standards.
Do not add any antigens in the
0 pg/ml (blank) point.
Streptavidin-PE incubated
too long
Follow the procedure incubation
time.
Poor Recovery
Expired Bio-Plex reagents were
used
Check that reagents have not
expired. Use new or unexpired
components.
Incorrect amounts of components
were added
Check your calculations and be
careful to add the correct volumes.
Microplate shaker set to an
incorrect speed
Check the microplate shaker speed
and use the recommended setting.
Setting the speed too high may
cause splashing and contamination.
Use the recommended plate shaker.
Pipetting technique
Pipet carefully when adding
standards, samples, detection
antibodies, and streptavidin-PE,
especially when using a
multichannel pipet. Use a calibrated
pipet. Change pipet tip after every
volume transfer.
27
Section 12
Safety Considerations
Eye protection and gloves are recommended while using this product.
Consult the MSDS for additional information.
The Bio-Plex Pro™ angiogenesis assays contain components of animal
origin. This material should be handled as if capable of transmitting
infectious agents. Please use universal precautions. These components
should be handled at Biosafety Level 2 containment [US Government
publication: Biosafety in Microbiological and Biomedical Laboratories
(CDC, 1999)].
28
xMAP is a trademark of Luminex Corp.
Costar is a trademark of Coming Costar Corporation. Eppendorf is a trademark of
Eppendorf-Netheler-Hinz GmbH. Luminex 100, xPONENET, and xMAP are trademarks of
Luminex Corporation. Multiscreen is a trademark of Millipore Corporation. Vortex-Genie is a
trademark
of Scientific Industries, Inc.
By purchasing this kit, which contains fluorescent labeled microsphere beads authorized by
Luminex, you, the customer, acquire the right under Luminex's patent rights* to use this kit or
any portion of this kit, including without limitation the microsphere beads contained herein, only
with Luminex’s laser-based fluorescent analytical test instrumentation known under the name
of Luminex 100, for example as marketed by Bio-Rad Laboratories, Inc. in the Bio-Plex
system.
*Including, but not limited to US patent 5,981,180; 6,046,807; 6,057,107.
31
Bio-Rad Laboratories, Inc.
2000 Alfred Nobel Dr.
Hercules, CA 94547 USA
1-800-424-6723 (in the US)
Bio-Rad
Laboratories, Inc.
Web site www.bio-rad.com USA 800 4BIORAD Australia 02 9914 2800
Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 21 3237 9400
Canada 905 712 2771 China 86 21 6426 0808
Czech Republic 420 241 430 532 Denmark 44 52 10 00
Finland 09 804 22 00 France 01 47 95 69 65 Germany 089 318 84 0
Greece 30 210 777 4396 Hong Kong 852 2789 3300
Hungary 36 1 455 8800 India 91 124 4029300/5013478 Israel 03 963 6050
Italy 39 02 216091 Japan 03 5811 6270 Korea 82 2 3473 4460
Mexico 55 5200 05 20 The Netherlands 0318 540666
New Zealand 64 9415 2280 Norway 23 38 41 30 Poland 48 22 331 99 99
Portugal 351 21 472 7700 Russia 7 095 721 14 04
Singapore 65 6415 3188 South Africa 27 0861 246 723
Spain 34 91 590 5200 Sweden 08 555 12700 Switzerland 061 717 95 55
Taiwan 886 2 2578 7189/2578 7241 United Kingdom 020 8328 2000
Life Science
Group
10010584
US/EG
Rev A
Sig 1106
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