CBA说明书

BD Brand

?Title BD ? Cytometric Bead Array (CBA)Mouse Th1/Th2 Cytokine Kit Cat. No. 551287

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BD flow cytometers are class I (1) laser products

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For research use only. Not for use in diagnostic or therapeutic procedures. Purchase does not include or carry any right to resell or transfer this product either as a stand-alone product or as a component of another product. Any use of this product other than the permitted use without the express written authorization of Becton Dickinson and Company is strictly prohibited.

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US Orders: 877.232.8995Kit Contents

80 Tests (50 Samples and 2 standard curves)

(Store the following items at 4°C)

A1 Mouse IL-2 Capture Beads: 1 vial, 0.8 ml

A2 Mouse IL-4 Capture Beads: 1 vial, 0.8 ml

A3 Mouse IL-5 Capture Beads: 1 vial, 0.8 ml

A4 Mouse IFN-γ Capture Beads: 1 vial, 0.8 ml

A5 Mouse TNF Capture Beads: 1 vial, 0.8 ml

B Mouse Th1/Th2 PE Detection Reagent: 1 vial, 4 ml

C Mouse Th1/Th2 Cytokine Standards: 2 vials, 0.2 ml lyophilized

D Cytometer Setup Beads: 1 vial, 1.5 ml

E1 PE Positive Control Detector: 1 vial, 0.5 ml

E2 FITC Positive Control Detector: 1 vial, 0.5 ml

F Wash Buffer: 1 bottle, 130 ml

G Assay Diluent: 1 bottle, 30 ml

Unless otherwise specified, all products are for Research Use Only.

Not for use in diagnostic or therapeutic procedures. Not for https://www.wendangku.net/doc/4b3549885.html, Table of Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5Principle of the Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6

Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6

Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7Reagents Provided . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7

Bead Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7

Antibody and Standard Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8

Buffer Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8 Warnings and Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8Materials Required but Not Provided . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9Overview: Mouse Th1/Th2 Cytokine CBA Assay Procedure . . . . . . . . . . . . . . .9Preparation of Mouse Th1/Th2 Cytokine Standards . . . . . . . . . . . . . . . . . . . .10Preparation of Mixed Mouse Th1/Th2 Cytokine Capture Beads . . . . . . . . . . .11Preparation of Test Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12Mouse Th1/Th2 Cytokine CBA Assay Procedure . . . . . . . . . . . . . . . . . . . . . .12Cytometer Setup, Data Acquisition and Analysis . . . . . . . . . . . . . . . . . . . . . . .13

Preparation of Cytometer Setup Beads . . . . . . . . . . . . . . . . . . . . . . . . . . .13 Instrument Setup with BD FACSComp Software

and BD Calibrite Beads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14

Instrument Setup with the Cytometer Setup Beads . . . . . . . . . . . . . . . . . .14

Data Acquisition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17 Analysis of Sample Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19Typical Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21

Theoretical Limit of Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21

Recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22

Linearity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23

Specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24 Precision . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25Troubleshooting Tips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27

Unless otherwise specified, all products are for Research Use Only.

Not for use in diagnostic or therapeutic procedures. Not for resale.5

US Orders: 877.232.8995Introduction

Flow cytometry is an analysis tool that allows for the discrimination of different particles on the basis of size and color . Multiplexing is the simultaneous assay of many analytes in a single sample . The BD? Cytometric Bead Array (CBA) employs a series of particles with discrete fluorescence intensities to simultaneously detect multiple soluble analytes . The BD CBA is combined with flow cytometry to create a powerful multiplexed assay .

The BD CBA system uses the sensitivity of amplified fluorescence detection by flow cytometry to measure soluble analytes in a particle-based immunoassay . Each bead in a BD CBA Kit provides a capture surface for a specific protein and is analogous to an individually coated well in an ELISA plate . The BD CBA capture bead mixture is in suspension to allow for the detection of multiple analytes in a small sample volume . The combined advantages of the broad dynamic range of fluorescent detection via flow cytometry and the efficient capturing of analytes via suspended particles enable the BD CBA to use fewer sample dilutions and to obtain the value of an unknown in substantially less time (compared to conventional ELISA) .

The BD Mouse Th1/Th2 Cytokine CBA Kit can be used to measure Interleukin-2 (IL-2), Interleukin-4 (IL-4), Interleukin-5 (IL-5), Interferon-γ (IFN-γ), and Tumor Necrosis Factor (TNF) protein levels in a single sample . The kit performance has been optimized for analysis of physiologically relevant concentrations (pg/ml levels) of specific cytokine proteins in tissue culture supernatants and serum samples .

Unless otherwise specified, all products are for Research Use Only.

Not for use in diagnostic or therapeutic procedures. Not for https://www.wendangku.net/doc/4b3549885.html, Principle of the Test

Five bead populations with distinct fluorescence intensities have been coated with capture antibodies specific for IL-2, IL-4, IL-5, IFN-γ, and TNF proteins . The five bead populations are mixed together to form the BD CBA, which is resolved in a red channel (ie, FL3 or FL4) of a flow cytometer .

IL-2

IL-4IL-5

IFN-γ

TNF Figure 1

The cytokine capture beads are mixed with the PE-conjugated detection antibodies and then incubated with recombinant standards or test samples to form sandwich complexes . Following acquisition of sample data using the flow cytometer , the sample results are generated in graphical and tabular format using the BD CBA Analysis Software or FCAP Array? Software . The kit provides sufficient reagents for the quantitative analysis of 50 test samples and the generation of two standard curve sets .

Advantages

The BD CBA provides several advantages when compared with conventional ELISA methodology:

? The required sample volume is approximately one-fifth the quantity necessary for conventional ELISA assays due to the detection of five analytes in a single sample .

? A single set of diluted standards is used to generate a standard curve for each analyte .

? A BD CBA experiment takes less time than a single ELISA and provides results that would normally require five conventional ELISAs

Unless otherwise specified, all products are for Research Use Only.

Not for use in diagnostic or therapeutic procedures. Not for https://www.wendangku.net/doc/4b3549885.html, Antibody and Standard Reagents

Mouse Th1/Th2 PE Detection Reagent (B): An 80-test vial of PE-conjugated anti-mouse IL-2, IL-4, IL-5, IFN-γ, and TNF antibodies that is formulated for use at 50 μl/test . Store at 4°C . Do not freeze .

PE Positive Control Detector (E1): A 10-test vial of PE-conjugated antibody control that is formulated for use at 50 μl/test . This reagent is used with the Cytometer Setup Beads to set the initial instrument compensation settings . Store at 4°C . Do not freeze .

FITC Positive Control Detector (E2): A 10-test vial of FITC-conjugated antibody control that is formulated for use at 50 μl/test . This reagent is used with the Cytometer Setup Beads to set the initial instrument compensation settings . Store at 4°C . Do not freeze .

Mouse Th1/Th2 Cytokine Standards (C): Two vials containing lyophilized recombinant mouse cytokine proteins . Each vial should be reconstituted in 2 .0 ml of Assay Diluent to prepare the top standard . Store at 4°C .

Buffer Reagents

Assay Diluent (G): A single 30 ml bottle of a buffered protein* solution (1×) used to reconstitute and dilute the Mouse Th1/Th2 Cytokine Standards and to dilute test samples . Store at 4°C .

Wash Buffer (F): A single 130 ml bottle of phosphate buffered saline (PBS) solution (1×), containing protein* and detergent, used for wash steps and to resuspend the washed beads for analysis . Store at 4°C .

Warnings and Precautions

Hazardous Ingredients:

Sodium Azide:

Components A1-A5, B, D, E1-E2, F , and G contain 0 .09%

sodium azide . Sodium azide yields a highly toxic hydrazoic

acid under acidic conditions . Avoid exposure to skin and eyes,

ingestion, and contact with heat, acids, and metals . Wash

exposed skin with soap and water . Flush eyes with water . Dilute

azide compounds in running water before discarding to avoid

accumulation of potentially explosive deposits in plumbing .

* Source of all serum proteins is from USDA inspected abattoirs located in the United States.

Unless otherwise specified, all products are for Research Use Only.

Not for use in diagnostic or therapeutic procedures. Not for resale.9

US Orders: 877.232.8995

Unless otherwise specified, all products are for Research Use Only.

Not for use in diagnostic or therapeutic procedures. Not for resale.

https://www.wendangku.net/doc/4b3549885.html,

Unless otherwise specified, all products are for Research Use Only.

Not for use in diagnostic or therapeutic procedures. Not for resale.11

US Orders: 877.232.8995

Unless otherwise specified, all products are for Research Use Only.

Not for use in diagnostic or therapeutic procedures. Not for https://www.wendangku.net/doc/4b3549885.html, Preparation of Test Samples

The standard curve for each cytokine covers a defined set of concentrations from 20 – 5000 pg/ml . It may be necessary to dilute test samples to ensure that their mean fluorescence values fall within the limits or range of the generated cytokine standard curve . For best results, samples that are known or assumed to contain high levels of a given cytokine should be diluted as described below .

1 . Dilute test sample by the desired dilution factor (ie, 1:2, 1:10 or 1:100) using the appropriate volume of Assay Diluent .

2 . Mix sample dilutions thoroughly before transferring samples to the appropriate assay tubes containing mixed Capture Beads and PE Detection Reagent .

BD CBA Mouse Th1/Th2 Cytokine Assay Procedure Following the preparation and dilution of the standards and mixing of the capture beads, transfer these reagents and test samples to the appropriate assay tubes for incubation and analysis . In order to calibrate the flow cytometer and quantitate test samples, it is necessary to run the Cytokine Standards and the Cytometer Setup controls in each experiment. See Table 2 for a detailed description of the reagents added to the Cytokine Standard control assay tubes . The Cytometer Setup procedure is described in Cytometer Setup, Data Acquisition and Analysis , page 13 .

1 . Add 50 μl of the mixed Capture Beads to the appropriate assay tubes . Vortex the mixed Capture Beads before adding to the assay tubes .

2 . Add 50 μl of the Mouse Th1/Th2 Cytokine Standard dilutions to the control assay tubes .

3 . Add 50 μl of each test sample to the test assay tubes .

4 . Add 50 μl of the Mouse Th1/Th2 PE Detection Reagent to the assay tubes .

5 . Incubate the assay tubes for 2 hours at RT and protect from direct exposure to light .

6 . Add 1 ml of Wash Buffer to each assay tube and centrifuge at 200 × g for 5 minutes .

7 . Carefully aspirate and discard the supernatant from each assay tube .

8 . Add 300 μl of Wash Buffer to each assay tube to resuspend the bead pellet . 9 . Begin analyzing samples on a flow cytometer . Vortex each sample for 3 - 5 seconds immediately before analyzing on the flow cytometer.

Note : It is necessary to analyze CBA samples on the day of the

experiment . Prolonged storage of samples, once the assay

is complete, can lead to increased background and reduced

sensitivity .

Unless otherwise specified, all products are for Research Use Only.

Not for use in diagnostic or therapeutic procedures. Not for resale.13

US Orders:

877.232.8995

Unless otherwise specified, all products are for Research Use Only.

Not for use in diagnostic or therapeutic procedures. Not for resale.

https://www.wendangku.net/doc/4b3549885.html, Instrument Setup with BD FACSComp Software

and BD Calibrite Beads

1 . Perform instrument start up .

2 . Perform flow check .

3 . Prepare tubes of BD Calibrite beads and open BD FACSComp software .

4 . Launch BD FACSComp software

5 . Run BD FACSComp software in Lyse/No Wash mode .

6 . Proceed to next section .

Note: For detailed information on using BD FACSComp with

BD Calibrite beads to set up the flow cytometer, refer to the

BD FACSComp Software User’s Guide and the BD Calibrite

Beads Package Insert . Version 4 .2 contains a BD CBA preference

setting to automatically save a BD CBA calibration file at

the successful completion of any Lyse/No Wash assay . The

BD CBA calibration file provides the optimization for FSC, SSC,

and threshold settings as described in Instrument Setup with

the Cytometer Setup Beads , Steps 3 – 5 . Optimization of the

fluorescence parameter settings is still required (ie, PMT and

compensation settings, see Instrument Setup with the Cytometer

Setup Beads , Step 6) .

Instrument Setup with the Cytometer Setup Beads

1 . Launch BD CellQuest Software and open the BD CBA Instrument Setup template .

Note: The BD CBA Instrument Setup template can be found on the

BD FACStation CD for Macintosh computers in the BD CBA

folder . This file may also be downloaded via the internet from: https://www.wendangku.net/doc/4b3549885.html,/cbatemplates

2 . Set the instrument to Acquisition mode .

Note : The data will be evaluated in five parameters (FSC, SSC, FL1,

FL2 and FL3) . Turn off additional detectors .

3 . Set SSC (side light scatter) and FSC (forward light scatter) to Log mode .

4 . Decrease the SSC PMT voltage by 100 from what BD FACSComp set .

5 . Set the Threshold to FSC at 650 .

6 . In setup mode, run Cytometer Setup Beads tube A . Follow the setup instructions on pages 15 – 1

7 .

Note : Pause and restart acquisition frequently during the instrument

setup procedure in order to reset detected values after settings

adjustments .

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Not for use in diagnostic or therapeutic procedures. Not for resale.15

US Orders: 877.232.8995010101010R1

100101102

103104

10R2

R3

Unless otherwise specified, all products are for Research Use Only.

Not for use in diagnostic or therapeutic procedures. Not for resale.17

US Orders: 877.232.8995Data Acquisition

1 . Open the acquisition template .

Note: The acquisition template may be downloaded via the internet

from: https://www.wendangku.net/doc/4b3549885.html,/cbatemplates

2 . Set acquisition mode and retrieve the optimized instrument settings from Instrument Setup with the Cytometer Setup Beads , page 14 .

3 . In the Acquisition and Storage window, set the resolution to 102

4 .

4 . Set number of events to be counted at 1500 of R1 gated events . (This will ensure that the sample file contains approximately 300 events per Capture Bead) .

5 . Set number of events to be collected to “all events” . Saving all events collected will ensure that no true bead events are lost due to incorrect gating .

6 . In setup mode, run tube no . 1 and using the FSC vs . SSC dot plot, place the R1 region gate around the singlet bead population (see Figure 3a ) .

7 . Samples are now ready to be acquired .

8 . Begin sample acquisition with the flow rate set at HIGH .

Note : Run the negative control tube (0 pg/ml standards) before any

of the recombinant standard tubes . Run the control assay tubes

before any unknown test assay tubes . Run the tubes in the order

listed in Table 2, page 12 .

File names must be alphanumeric (ie, contain at least one letter) .

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Not for use in diagnostic or therapeutic procedures. Not for resale.

https://www.wendangku.net/doc/4b3549885.html, Figure 4. Acquisition Template Example

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US Orders: 877.232.8995104321

04321

104

10321

0104

321

Unless otherwise specified, all products are for Research Use Only.

Not for use in diagnostic or therapeutic procedures. Not for resale.

https://www.wendangku.net/doc/4b3549885.html,