Bio-Rad Human MMP and TIMP Assays User Manual

Bio-Plex Pro

™

Human

TIMP Assays

Instruction Manual

For technical support, call your local Bio-Rad ofﬁce, or in the U.S., call 1-800-424-6723. For research use only. Not for diagnostic procedures.

Table of Contents

Introduction 1

Principle 2

Kit Contents and Storage 4

Recommended Materials 5

Assay Workﬂow 6

lmportant Considerations 7

Detailed Instructions 7

1.1. Plan Plate Layout 8

2.2. Prepare Instrument 9

3.3. Prepare Wash Method 10

4.4. Prepare Wash Buffer 11

5.5. Prepare Standards and Controls 11

6. Prepare Samples 14

7.7. Prepare Coupled Beads 16

8.8. Run Assay 17

9.9. Read Plate 21

Troubleshooting Guide 28

Plate Layout Template 33

Calculation Worksheet 34

Safety Considerations 35

Legal Notices 35

Ordering Information 36

Introduction

Bio-Plex Pro™ Human TIMP Assays

The matrix metalloproteinases (MMPs) are a family of zinc proteases with essential roles in breaking down components of the extracellular matrix (ECM). The MMPs are inhibited by speciﬁc endogenous tissue inhibitors of metalloproteinases (TIMPs), which are a family of four protease inhibitors: TIMP-1, TIMP-2, TIMP-3, and TIMP-4. The balance between MMP and TIMP levels is crucial for the timely degradation of ECM.

MMPs and TIMPs play important roles in tissue remodeling associated with various physiological and pathological processes such as inﬂammation, apoptosis, morphogenesis, angiogenesis, tumor invasion, and metastasis. The new MMP and TIMP panels will expand the Bio-Plex Pro assay menu in research areas including cancer, autoimmune disease, wound healing, and cardiovascular disease.

Advantages of Magnetic Bead-Based Assays

Products in the Bio-Plex Pro family of assays are on magnetic polystyrene beads. These beads provide a choice in the method of assay preparation — standard or magnet-based. The standard workﬂow for Bio-Plex assay preparation requires multiple wash steps in which the 96-well ﬁlter plate is placed on a vacuum manifold to draw the liquid through the bottom of the ﬁlter plate. In contrast, magnet-based assay preparation permits liquid removal from the top of the well and thus does not require a ﬁlter plate or vacuum manifold. As a result, either an automated or manual washer can be used. Bio-Rad offers both solutions with the automated Bio-Plex Pro wash station and the manual Bio-Plex handheld magnetic washer. Magnetic separation offers greater convenience and reproducibility compared to vacuum ﬁltration.

For a current listing of Bio-Plex assays, panels, and reagents, please visit www.bio-rad.com/bio-plex.

Principle

Technology

The Bio-Plex

suspension array system is built upon the three core

elements of xMAP technology:

Fluorescently dyed microspheres (also called beads), each with a distinct

color code or spectral address to permit discrimination of individual tests within a multiplex suspension. This allows simultaneous detection of more than 100 different types of molecules in a single well of a 96-well microplate

A dedicated ﬂow cytometer with two lasers and associated optics to

measure the different molecules bound to the surface of the beads. In the Bio-Plex

MAGPIX™ system, the sample is injected into a chamber

where the beads are imaged using LED and CCD technology

A high-speed digital signal processor that efﬁciently manages the

ﬂuorescence data

Assay Format

Bio-Plex Pro

™

assays are essentially immunoassays formatted on magnetic beads. The assay principle is similar to that of a sandwich ELISA (Figure 1). Capture antibodies directed against the desired biomarker are covalently coupled to the beads. Coupled beads react with the sample containing the biomarker of interest. After a series of washes to remove unbound protein, a biotinylated detection antibody is added to create a sandwich complex. The ﬁnal detection complex is formed with the addition of streptavidin-phycoerythrin (streptavidin-PE or SA-PE) conjugate. Phycoerythrin serves as a ﬂuorescent indicator, or reporter.

Biomarker of Interest

Streptavidin

Magnetic Bead

Capture

Antibody

Fig. 1. Bio-Plex sandwich immunoassay.

Biotinylated

Detection

Antibody

Phycoerythrin

Fluorescent

Reporter

Data Acquisition and Analysis

Data from the reactions are acquired using a Bio-Plex system or similar Luminex-based reader. When a multiplex assay suspension is drawn into the Bio-Plex 200 reader, for example, a red (635 nm) laser illuminates the ﬂuorescent dyes within each bead to provide bead classiﬁcation and thus assay identiﬁcation. At the same time, a green (532 nm) laser excites PE to generate a reporter signal, which is detected by a photomultiplier tube (PMT). A high-speed digital processor manages data output, and Bio-Plex Manager

™

software presents data as median ﬂuorescence intensity (MFI) as well as concentration. The concentration of analyte bound to each bead is proportional to the MFI of the reporter signal.

Kit Contents and Storage

Reagents Supplied

Bio-Plex Pro

™

TIMP assays are available in a convenient all-in-one kit format

that includes assay, reagent, and diluent components in a single box.

Table 1. Contents of 1 x 96-well kits.

Component

Diluent HD

Assay buffer

Wash buffer (10x)

Detection antibody diluent HB

Streptavidin-PE (100x)

Assay plate (96-well ﬂat bottom plate)

Sealing tape

Assay quick guide

Product data sheet

Coupled magnetic beads (20x)

Detection antibodies (20x)

Standard

Control

* Volumes shown are approximate.

1 bottle, 180 ml

1 bottle, 50 ml

1 bottle, 60 ml

1 bottle, 5 ml

Quantity

1 tube

1 plate

1 pack of 4

1 booklet

1 tube

1 vial

Storage and Stability

Kit contents should be stored at 4°C and never frozen. Coupled magnetic beads and streptavidin-PE should be stored in the dark. All components are guaranteed for a minimum of six months from the date of purchase when stored as speciﬁed.

Table 2. Recommended materials.

Item

Bio-Plex® 200 system or Luminex system with HTF

Bio-Plex validation kit

Ordering Information

Bio-Rad catalog #171-000205

Bio-Rad catalog #171-203001 Note: Run the validation kit monthly to ensure optimal performance of ﬂuidics and optics systems

Bio-Plex calibration kit

Bio-Rad catalog #171-203060 Note: Run the calibration kit daily to standardize ﬂuorescence signal

Bio-Plex Pro wash station

Bio-Rad catalog #300-34376 For use with magnetic bead–based assays only

Bio-Plex handheld magnetic washer

Bio-Rad catalog #170-20100 For use with magnetic bead–based assays only

Bio-Plex Pro ﬂat bottom plates (forty 96-well plates)

Bio-Rad catalog #171-025001 For magnetic separation on the Bio-Plex Pro wash station

Titertube

For preparing replicate standards, samples, and controls

micro test tubes

Bio-Rad catalog #223-9390 prior to loading the plate

Microtiter plate shaker

IKA MTS 2/4 shaker for 2 or 4 microplates

IKA catalog #320-8000 or Barnstead/Lab-Line Model 4625 plate

VWR catalog #57019-600 shaker (or equivalent capable of 300–1,100 rpm)

Bio-Rad

Aurum™ vacuum manifold

Bio-Rad catalog #732-6470 For vacuum ﬁltration

BR-2000 vortexer

Reagent reservoirs, 25 ml

For capture beads and detection antibodies

Reagent reservoir, 50 ml (for reagents and buffers)

Pall Life Science Acrodisc: 25 mm PF syringe ﬁlter

(0.8/0.2 µm Supor membrane)

Filter plate, 1 x 96 with clear plastic lid and tray

™

Bio-Plex Manager

MP Software Upgrade

Bio-Rad catalog #166-0610

VistaLab catalog #3054-1002

VistaLab catalog #3054-1004

VistaLab catalog #3054-1006

Pall Life Sciences

catalog #4187

Bio-Rad catalog #171-304502

Bio-Rad catalog #171-051555

Other: 15 ml polypropylene tubes for reagent dilutions, calibrated pipets, pipet tips, sterile distilled water, aluminum foil, absorbent paper towels, 1.5 or 2 ml microcentrifuge tubes, and standard ﬂat bottom microplate (for calibrating vacuum manifold).

Assay Workﬂow

Prewet wells

(for lter plate only)

Add 50 µl 1x beads to wells

Wash 2 x 100 μl

Add 50 μl diluted standards, samples, controls,

incubate 1 hr at RT with shaking at 850 rpm

Wash 3 x 100 μl

Add 25 μl 1x detection antibody, incubate

30 min at RT with shaking at 850 rpm

Wash 3 x 100 μl

Add 50 μl 1x streptavidin-PE, incubate

10 min at RT with shaking at 850 rpm

Wash 3 x 100 μl

Resuspend in 125 μl assay buffer,

shake at 850 rpm for 30 sec

Acquire data on Bio-Plex system

lmportant Considerations

Instruments and Software

The Bio-Plex Pro

™

assays described in this manual are compatible with all currently available Luminex-based life science research instruments. Assays can be read and analyzed with either Bio-Plex Manager

™

software

or Luminex xPONENT software (section 9).

Assay Procedures

Pay close attention to vortexing, shaking, and incubation times and to Bio-Plex

reader PMT (RP1) setting, as these have been optimized

speciﬁcally for each assay panel.

Assay Quick Guide

Each assay kit includes a printed Bio-Plex Pro Assay Quick Guide (bulletin #10041638), which can be used to prepare and run a full 1 x 96-well assay plate. Users can also download a copy at www.bio-rad.com/bio-plex.

Bead Regions

Bead regions for all analytes are listed in the Read Plate section.

Detailed Instructions

The following pages provide detailed instructions for each step of the assay procedure, including preparation, running the assay, and reading the plate with Bio-Plex Manager

™

and Luminex xPONENT software.

1. Plan Plate Layout

Prior to running the assay, determine the total number of wells in the experiment using the Plate Layout Template on page 33 or the Plate Formatting tab in Bio-Plex Manager is shown in Figure 2, with all conditions in duplicate.

1. Assign standards to columns 1 and 2, with the highest concentration

in row A and the lowest concentration in row H.

2. Assign the blank to wells A3 and A4. The blank should consist of your

chosen diluent HD or a diluent similar to your ﬁnal sample type or matrix. Note that Bio-Plex Manager automatically subtracts the blank (B) MFI value from all other assay wells.

3. Controls, either user-speciﬁed or the controls supplied, are assigned to

wells in columns 3 and 4.

4. The remainder of the plate is available for samples.

5. Once the total number of wells is known, calculate the required volumes

of beads, detection antibody, and streptavidin-PE needed. Use Tables 6, 8, and 9, respectively, or the Calculation Worksheet on page 34.

Legend

S Standard

™

software. A suggested plate layout

B Blank

X Samples

C Controls

Fig. 2. Suggested plate layout. For detailed instructions on plate formatting in Bio-Plex Manager software, see section Read Plate.

2. Prepare Instrument

These directions are speciﬁc for the Bio-Plex® 100/200 reader. To prepare either a Bio-Plex 3D or Bio-Plex user manuals.

Note: While the instrument is warming up, bring the 10x wash buffer, assay buffer, and diluent HD to room temperature. Keep other items on ice until needed. Also, begin to thaw frozen samples.

Start up and calibrate the Bio-Plex system with Bio-Plex Manager software prior to setting up the assay. The calibration kit should be run daily or before each use of the instrument to standardize the ﬂuorescent signal. For instructions on using other xMAP system software packages, contact Bio-Rad Technical Support.

The validation kit should be run monthly to ensure optimal performance of ﬂuidics and optics systems. Refer to either the software manual or online Help for directions on how to conduct validation.

MAGPIX™ reader, consult their respective

™

Start Up System (Bio-Plex 100, 200, or similar)

1. Empty the waste bottle and ﬁll the sheath ﬂuid bottle before starting

if high throughput ﬂuidics (HTF) are 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 (if not already done).

3. Select Start up

for 4 hr without acquiring data, the lasers will automatically turn off. To reset the 4-hr countdown, select Warm up lasers/optics to reach operational temperature.

and follow the instructions. If the system is idle

and wait for the

Calibrate System

1. Select Calibrate and conﬁrm that the default values for CAL1

and CAL2 are the same as the values printed on the bottle of Bio-Plex calibration beads. Use the Bio-Plex system low RP1 target value.

2. Select OK and follow the software prompts for step-by-step

instructions for CAL1 and CAL2 calibration.

Note: In Bio-Plex Manager version 6.1 and higher, startup, warm up,

and calibration can be performed together by selecting the “Start up and calibrate” icon.

3. Prepare Wash Method

Bio-Plex Pro assays are compatible with both magnetic separation and vacuum ﬁltration methods. However, for best results, we recommend performing the assays in a ﬂat bottom plate with magnetic separation.

Table 3. Summary of compatible wash stations and plate types.

Wash Method Wash Station Assay Plate

Magnetic separation Bio-Plex Pro Bio-Plex handheld magnetic washer

Vacuum ﬁltration Vacuum manifold (manual) Filter plate

Setting Up the Bio-Plex Pro Wash Station

The wash station does not require calibration; however, it should be primed before use. For more information, refer to the Bio-Plex Pro wash station quick guide (bulletin #5826).

1. Install the appropriate plate carrier on the wash station.

2. Use the prime procedure to prime channel 1 with wash buffer.

™

Flat bottom plate

Setting Up the Bio-Plex Handheld Magnetic Washer

Place an empty ﬂat bottom plate on the magnetic washer by sliding it under the retaining clips. Push the clips inward to secure the plate. Make sure the plate is held securely. If needed, the clips can be adjusted for height and tension. For detailed instructions, refer to the user guide (bulletin #10023087).

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