BD Citocinas

BD Cytometric Bead Array
(CBA) Human Inflammatory

Cytokines Kit
Instruction Manual
Catalog No. 551811

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Contents
Chapter 1: About this kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Purpose of the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Kit contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Storage and handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Chapter 2: Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Workflow overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Chapter 3: Assay preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Preparing Human Inflammatory Cytokines Standards . . . . . . . . . . . 18
Mixing Human Inflammatory Cytokine Capture Beads . . . . . . . . . . 20
Diluting samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Chapter 4: Assay procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Performing the Human Inflammatory Cytokine Assay . . . . . . . . . . . 24
Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Chapter 5: Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Theoretical limit of detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Linearity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Precision . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Chapter 6: Reference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
1
About this kit
This section covers the following topics:
 Purpose of the kit (page 6)

 Limitations (page 8)
 Kit contents (page 9)
 Storage and handling (page 12)
6 BD CBA Human Inflammatory Cytokines Kit
Purpose of the kit

Use of the kit The BD™ CBA Human Inflammatory Cytokines Kit can
be used to quantitatively measure interleukin-8 (IL-8),
interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-
10 (IL-10), tumor necrosis factor (TNF), and interleukin-
12p70 (IL-12p70) protein levels in a single sample. The
kit performance has been optimized for analysis of
specific proteins in tissue culture supernatants, EDTA-
treated plasma, and serum samples using one of two
protocols, depending on the sample source. The kit
provides sufficient reagents for 80 tests.
Principle of CBA BD CBA assays provide a method of capturing a soluble

assays analyte or set of analytes with beads of known size and
fluorescence, making it possible to detect analytes using
flow cytometry.
Each capture bead in the kit has been conjugated with a

specific antibody. The detection reagent provided in the
kit is a mixture of phycoerythrin (PE)–conjugated
antibodies, which provides a fluorescent signal in
proportion to the amount of bound analyte.
When the capture beads and detector reagent are

incubated with an unknown sample containing
recognized analytes, sandwich complexes (capture bead
+ analyte + detection reagent) are formed. These
complexes can be measured using flow cytometry to
identify particles with fluorescence characteristics of
both the bead and the detector.
Chapter 1: About this kit 7
Principle of this Six bead populations with distinct fluorescence

assay intensities have been coated with capture antibodies
specific for IL-8, IL-1, IL-6, IL-10, TNF, and IL-12p70
proteins. The six bead populations are mixed together to
form the bead array that is resolved in a red channel
(FL3 or FL4) of a flow cytometer (see Figure 1).
Figure 1
During the assay procedure, you will mix the

inflammatory cytokine capture beads with the
recombinant standards or unknown samples and
incubate them with the PE-conjugated detection
antibodies to form sandwich complexes. The intensity of
PE fluorescence of each sandwich complex reveals the
concentration of that cytokine. After acquiring samples
on a flow cytometer, use FCAP Array™ software to
generate results in graphical and tabular format.
Advantages over The broad dynamic range of fluorescent detection via

ELISA flow cytometry and the efficient capturing of analytes via
suspended particles enable BD CBA assays to measure
the concentration of an unknown in substantially less
time and using fewer sample dilutions compared to
conventional ELISA methodology.
 The required sample volume is approximately one-

sixth the quantity necessary for conventional ELISA
assays due to the detection of six 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 six conventional ELISAs.
Limitations
Assay limitations This kit is designed to be used as an integral unit. Do not
mix components from different batches or kits.
The theoretical limit of detection of the BD CBA Human

Inflammatory Cytokines Kit is comparable to
conventional ELISA, but due to the complexity and
kinetics of this multi-analyte assay, the actual limit of
detection in a given experiment may vary slightly. See
Theoretical limit of detection (page 35) and Precision
(page 41).
The BD CBA Kit is not recommended for use on stream-

in-air instruments for which signal intensities may be
reduced, adversely affecting assay sensitivity. Stream-in-
air instruments include the BD FACStar™ Plus,
BD FACSVantage™, and BD Influx™ flow cytometers
(BD Biosciences).
Serum spike recoveries for IL-1, TNF, and IL-12p70 are

lower than for the other proteins in this assay. This
variation is due to assay conditions and serum proteins
and may affect quantitation of these proteins in serum
samples.
The sensitivity for the detection of IL-1 in this assay is

less than for the other proteins measured. It is possible
that, due to operator variation, instrument settings, and
instrument performance, the 20 pg/mL standard curve
point for IL-1 may not have signal intensity above the
0 pg/mL background level.
Quantitative results or protein levels for the same sample

or recombinant protein run in ELISA and BD CBA
assays may differ. A spike recovery assay can be
performed using an ELISA standard followed by
BD CBA analysis to assess possible differences in
quantitation.
The BD CBA Human Inflammatory Cytokines Kit assay

has been shown to detect IL-8, IL-6, and TNF produced
by the activation of cells from the non-human primate
rhesus and cynomolgus models. Direct quantitation of
proteins from the rhesus and cynomolgus models has not
been validated using this kit, and results may vary.
Kit contents
Contents This kit contains the following components sufficient for
80 tests.
Vial
label Reagent Quantity
A1 Human IL-8 Capture Beads 1 vial, 0.8 mL
A2 Human IL-1 Capture Beads 1 vial, 0.8 mL
A5 Human TNF Capture Beads 1 vial, 0.8 mL
A6 Human IL-12p70 Capture Beads 1 vial, 0.8 mL
Vial
label Reagent Quantity
B Human Inflammatory Cytokine 1 vial, 4 mL
PE Detection Reagent
C Human Inflammatory Cytokine 2 vials
Standards 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, 260 mL
G Assay Diluent 1 bottle, 30 mL
H Serum Enhancement Buffer 1 bottle, 10 mL
Bead reagents Human Inflammatory Cytokine Capture Beads (A1–

A6): An 80-test vial of each specific capture bead (A1–
A6). The specific capture beads, having discrete
fluorescence intensity characteristics, are distributed
from brightest (A1) to dimmest (A6).
Cytometer Setup Beads (D): A 30-test vial of setup beads

for setting the initial instrument PMT voltages and
compensation settings is sufficient for 10 instrument
setup procedures. The Cytometer Setup Beads are
formulated for use at 50 µL per test.
Antibody and Human Inflammatory Cytokine PE Detection Reagent

standard (B): An 80-test vial of PE-conjugated anti-human IL-8,
reagents IL-1ß, IL-6, IL-10, TNF, and IL-12p70 antibodies,
formulated for use at 50 µL per test.
Human Inflammatory Cytokine Standards (C): Two

vials containing lyophilized recombinant human
proteins. Each vial should be reconstituted in 2.0 mL of
Assay Diluent to prepare the top standard.
PE Positive Control Detector (E1): A 10-test vial of

PE-conjugated antibody control formulated for use at
50 µL per test. This reagent is used with the Cytometer
Setup Beads to set the initial instrument compensation
settings.
FITC Positive Control Detector (E2): A 10-test vial of

FITC-conjugated antibody control formulated for use at
50 µL per test. This reagent is used with the Cytometer
Setup Beads to set the initial instrument compensation
settings.
Buffer reagents Wash Buffer (F): A 260-mL bottle of phosphate buffered

saline (PBS) solution (1X), containing protein and
detergent, used for wash steps and to resuspend the
washed beads for analysis.
Assay Diluent (G): A 30-mL bottle of a buffered protein

solution (1X) used to reconstitute and dilute the Human
Inflammatory Cytokine Standards and to dilute
unknown samples.
Serum Enhancement Buffer (H): A 10-mL bottle of a

buffered protein solution (1X) used to dilute mixed
Capture Beads when testing serum or plasma samples.
Note: Source of all serum proteins is from USDA-

inspected abattoirs located in the United States.
Storage and handling

Storage Store all kit components at 2 to 8°C. Do not freeze.
Appearance: The visual appearance of Assay Diluent

may range in color from red to yellow/orange. The visual
appearance of ELISA Dilution reagent may range in
color from clear to cloudy white.
Warning Components A1–A6, B, D, E1–E2, F, G, and H contain

sodium azide. Sodium azide yields a highly toxic
hydrazoic acid under acidic conditions. Dilute azide
compounds in running water before discharging to avoid
accumulation of potentially explosive deposits in
plumbing.
Human Inflammatory Standards (component 51-

2553KC) contains 0.02% (w/w) of a CMIT/MIT
mixture (3:1), which is a mixture of: 5-chloro-2-methyl-
4-isothiazolin-3-one [EC No 247-500-7] and 2-methyl-
4-isothiazolin-3-one [EC No 220-239-6] (3:1).
Hazard May cause an allergic skin reaction.

statements
Precautionary Wear protective gloves / eye protection.

statements
Wear protective clothing.
Avoid breathing mist/vapours/spray.
If skin irritation or rash occurs: Get medical advice/

attention.
IF ON SKIN: Wash with plenty of water.
Dispose of contents/container in accordance with local/

regional/national/international regulations.
2
Before you begin
 Workflow overview (page 15)

 Required materials (page 16)
Workflow overview
Workflow The overall workflow consists of the following steps.
Step Description
1 Preparing Human Inflammatory Cytokines Standards
(page 19)
2 Mixing Human Inflammatory Cytokine Capture
Beads (page 21)
3 Diluting samples (page 23)
4 Performing instrument setup with Cytometer Setup
Beads (instructions can be found at
bdbiosciences.com/cbasetup)
Note: Can be performed during the incubation in
step 5.
5 Performing the Human Inflammatory Cytokine Assay
(page 25)
6 Acquiring samples (instructions can be found at
bdbiosciences.com/cbasetup)
7 Data analysis (page 31)
Incubation times To help you plan your work, the incubation times are
listed in the following table.
Procedure Incubation time

Preparing standards 15 minutes
Preparing mixed capture beads (when 30 minutes
analyzing serum or plasma samples only)
Preparing Cytometer Setup Beads 30 minutes
Performing the assay 3 hours
Chapter 2: Before you begin 16
Required materials
Materials In addition to the reagents provided in the BD CBA
required but not Human Inflammatory Cytokines Kit, the following items
provided are also required:
 A dual-laser flow cytometer equipped with a 488-

nm or 532-nm and a 633-nm or 635-nm laser
capable of distinguishing 576-nm, 660-nm, and
>680-nm fluorescence. The following table lists
examples of compatible instrument platforms.
Reporter
Flow cytometer channel Bead channels
BD FACSArray™ Yellow Red
BD FACSCanto™ platform
BD™ LSR platform
BD FACSAria™ platform PE APC
BD FACSCalibur™ (single laser) FL3
BD FACSCalibur (dual laser) FL2 FL4
Note: Visit bdbiosciences.com/cbasetup for setup protocols.
 Falcon® 12 × 75-mm sample acquisition tubes for a

flow cytometer (Catalog No. 352008)
 15-mL conical, polypropylene tubes (Falcon,
Catalog No. 352097), or equivalent
 FCAP Array software (Catalog No. 641488 [PC] or
645447 [Mac®])
Materials  Millipore MultiScreenHTS-BV 1.2 µm Clear non-

required for sterile filter plates [Catalog No. MSBVN1210 (10
plate loader- pack) or MSBVN1250 (50 pack)]
equipped flow
 Millipore MultiScreenHTS Vacuum Manifold,
cytometers
(Catalog No. MSVMHTS00)
 MTS 2/4 Digital Stirrer, IKA Works, VWR (Catalog
No. 82006-096)
 Vacuum source
 Vacuum gauge and regulator (if not using the
recommended manifold)
3
Assay preparation
 Preparing Human Inflammatory Cytokines Standards (page 19)

 Mixing Human Inflammatory Cytokine Capture Beads (page 21)
 Diluting samples (page 23)
Preparing Human Inflammatory Cytokines Standards

Purpose of this The Human Inflammatory Cytokines Standards are
procedure lyophilized and should be reconstituted and serially
diluted immediately before mixing with the Capture
Beads and the PE Detection Reagent.
You must prepare fresh standards to run with each

experiment. Do not store or reuse reconstituted or
diluted standards.
Procedure To reconstitute and serially dilute the standards:

1. Open one vial of lyophilized Human Inflammatory
Cytokine Standards. Transfer the standard spheres
to a 15-mL polypropylene tube. Label the tube “Top
Standard.”
2. Reconstitute the standards with 2 mL of Assay
Diluent.
a. Allow the reconstituted standard to equilibrate
for at least 15 minutes at room temperature.
b. Gently mix the reconstituted protein by pipette
only. Do not vortex or mix vigorously.
3. Label eight 12 × 75-mm tubes and arrange them in
the following order: 1:2, 1:4, 1:8, 1:16, 1:32, 1:64,
1:128, and 1:256.
4. Pipette 300 µL of Assay Diluent in each of the 12 ×
75-mm tubes.
5. Perform a serial dilution:
a. Transfer 300 µL from the Top Standard to the
1:2 dilution tube and mix thoroughly by pipette
only. Do not vortex.
Chapter 3: Assay preparation 20
b. Continue making serial dilutions by transferring

300 µL from the 1:2 tube to the 1:4 tube and so
on to the 1:256 tube.
6. Prepare one 12 × 75-mm tube containing only Assay

Diluent to serve as the 0 pg/mL negative control.
Concentration of See the Performing the Human Inflammatory Cytokine

standards Assay (page 25) for a listing of the concentrations (pg/
mL of all six recombinant proteins in each standard.
Next step Proceed to Mixing Human Inflammatory Cytokine

Capture Beads (page 21).
Mixing Human Inflammatory Cytokine Capture Beads

Purpose of this The Capture Beads are bottled individually (A1–A6).
procedure You must pool all six bead reagents immediately before
using them in the assay.
Mixing the beads To mix the Capture Beads:

1. Determine the number of assay tubes (including
standards and controls) that are required for the
experiment (for example, 8 unknowns + 9 standard
dilutions + 1 negative control = 18 assay tubes).
2. Vigorously vortex each Capture Bead suspension for
3 to 5 seconds before mixing.
Note: The antibody-conjugated beads will settle out
of suspension over time. Vortex the vial immediately
before taking a bead-suspension aliquot.
3. Add a 10-µL aliquot of each Capture Bead, for each

assay tube to be analyzed, into a single tube labeled
“Mixed Capture Beads” (eg, 10 µL of IL-8 Capture
Beads × 18 assay tubes = 180 µL of IL-8 Capture
Beads required).
4. Vortex the bead mixture thoroughly.
Resuspending If you are using serum or plasma samples, you must

the beads perform this procedure to reduce the chances of false-
positive results due to serum or plasma proteins. This
procedure is optional for all other sample types.
To resuspend the Capture Beads in Serum Enhancement

Buffer:
1. Centrifuge the mixed Capture Beads at 200g for
5 minutes.
2. Carefully aspirate and discard the supernatant.
Chapter 3: Assay preparation 22
3. Resuspend the mixed Capture Beads pellet in Serum

Enhancement Buffer (equal to the volume removed
in step 2) and vortex thoroughly.
4. Incubate the mixed Capture Beads for 30 minutes at
room temperature, protected from light.
Next step The mixed Capture Beads are now ready to be

transferred to the assay tubes. Discard excess mixed
Capture Beads. Do not store after mixing.
To begin the assay, proceed to Performing the
Human Inflammatory Cytokine Assay (page 25). If
you need to dilute samples having a high-protein
concentration, proceed to Diluting samples
(page 23).
Diluting samples
Purpose of this The standard curve for each protein covers a defined set
procedure of concentrations from 20 to 5,000 pg/mL. It might be
necessary to dilute test samples to ensure that their mean
fluorescence values fall within the range of the generated
standard curve. For best results, dilute samples that are
known or assumed to contain high levels of a given
protein. This procedure is not required for all samples.
Procedure To dilute samples with known high-cytokine

concentration:
1. Dilute the sample by the desired dilution factor (for
example, 1:2, 1:10, or 1:100) using the appropriate
volume of Assay Diluent.
Optimal recovery from serum samples typically
requires a 1:4 dilution.
2. Mix sample dilutions thoroughly.
Next step Perform instrument setup using the Cytometer Setup

Beads. For details, go to bdbiosciences.com/cbasetup and
select the appropriate flow cytometer under CBA Kits:
Instrument Setup.
Or, if you wish to begin staining your samples for the

assay, proceed to Performing the Human Inflammatory
Cytokine Assay (page 25), and you can perform
instrument setup during the 3-hour staining incubation.
4
Assay procedure
 Performing the Human Inflammatory Cytokine Assay (page 25)

 Data analysis (page 31)
Performing the Human Inflammatory Cytokine Assay

Before you begin 1. Prepare the standards as described in Preparing
Human Inflammatory Cytokines Standards
(page 19).
2. Mix the Capture Beads as described in Mixing
Human Inflammatory Cytokine Capture Beads
(page 21).
3. If necessary, dilute the unknown samples. See
Diluting samples (page 23).
Procedure for To perform the assay:

supernatant 1. Vortex the mixed Capture Beads and add 50 µL to
samples all assay tubes.
2. Add 50 µL of the Human Inflammatory Cytokine
Standard dilutions to the control tubes as listed in
the following table.
Concentration
Tube label (pg/mL) Standard dilution
1 0 (negative control) no standard dilution
(Assay Diluent only)
2 20 1:256
3 40 1:128
4 80 1:64
5 156 1:32
6 312.5 1:16
7 625 1:8
8 1,250 1:4
9 2,500 1:2
10 5,000 Top Standard
Chapter 4: Assay procedure 26
3. Add 50 µL of each unknown sample to the

appropriately labeled sample tubes.
4. Add 50 µL of the Human Inflammatory Cytokine PE
Detection Reagent to all assay tubes.
5. Incubate the assay tubes for 3 hours at room
temperature, protected from light.
Note: If you have not yet performed cytometer
setup, you may wish to do so during this incubation.
6. Add 1 mL of Wash Buffer to each assay tube and

centrifuge at 200g 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.

serum/plasma 1. Vortex the mixed Capture Beads and add 50 µL to
samples all assay tubes.
2. Add 50 µL of the Human Inflammatory Cytokine
Standard dilutions to the control tubes as listed in
the following table.
Concentration
1 0 (negative control) no standard dilution
(Assay Diluent only)
2 20 1:256
3 40 1:128
4 80 1:64
5 156 1:32
6 312.5 1:16
7 625 1:8
Concentration
8 1,250 1:4
9 2,500 1:2
10 5,000 Top Standard
3. Add 50 µL of each unknown sample to the

appropriately labeled sample tubes.
4. Incubate the assay tubes for 1.5 hours at room
Note: If you have not yet performed cytometer
setup, you may wish to do so during this incubation,
or during the incubation in step 8.

6. Carefully and consistently aspirate and discard the
supernatant, leaving approximately 100 µL of liquid
in each assay tube.
7. Add 50 µL of the Human Inflammatory Cytokine PE
Detection Reagent to all assay tubes. Gently agitate
the tubes to resuspend the pellet.
8. Incubate the assay tubes for 1.5 hours at room
10. Carefully aspirate and discard the supernatant from
each assay tube.
11. Add 300 µL of Wash Buffer to each assay tube to
resuspend the bead pellet.

filter plates for 1. Wet the plate by adding 100 µL of wash buffer to
supernatant each well.
samples
2. Place the plate on the vacuum manifold.
3. Aspirate for 2 to 10 seconds until the wells are
drained.
4. Remove the plate from the manifold, then blot the
bottom of the plate on paper towels.
5. Add 50 µL of each of the following to the wells in
the filter plate:
• Capture Beads (vortex before adding)
• Standard or sample (add standards from the
lowest concentration to the highest, followed by
samples)
• Human Inflammatory Cytokine PE Detection
Reagent
6. Cover the plate and shake it for 5 minutes at 1,100
rpm on a plate shaker.
7. Incubate the plate for 3 hours at room temperature
on a non-absorbent, dry surface.
Note: Place the plate on a non-absorbent, dry
surface during incubation. Absorbent or wet
surfaces can cause the contents of the wells to leak.
8. Remove the cover from the plate and apply the plate
to the vacuum manifold.
9. Vacuum aspirate for 2 to 10 seconds until the wells
are drained.
bottom of the plate on paper towels after aspiration.
11. Add 120 µL of wash buffer to each well to resuspend

the beads.
rpm before you begin sample acquisition.

filter plates for 1. Wet the plate by adding 100 µL of wash buffer to
serum/plasma each well.
samples
2. Place the plate on the vacuum manifold.
3. Aspirate for 2 to 10 seconds until the wells are
drained.
bottom of the plate on paper towels.
5. Add 50 µL of each of the following to the wells in
the filter plate:
• Capture Beads (vortex before adding)
• Standard or sample (add standards from the
lowest concentration to the highest, followed by
samples)
7. Incubate the plate for 1.5 hours at room temperature
8. Remove the cover from the plate and apply the plate
to the vacuum manifold.
9. Vacuum aspirate for 2 to 10 seconds until the wells
are drained.
bottom of the plate on paper towels after aspiration.
11. Add 200 µL of wash buffer to each well. Cover the
plate and shake for 2 minutes at 1,100 rpm.
12. Repeat step 8 through step 10.
13. Add 100 µL of assay diluent to each well.
14. Add 50 µL of Human Inflammatory Cytokine PE
Detection Reagent to each well.
16. Incubate the plate for 1.5 hours at room temperature
17. Repeat step 8 through step 10.

18. Add 120 µL of wash buffer to each well to resuspend
the beads.
19. Shake the plate for 2 minutes at 1,100 rpm before
you begin sample acquisition.
Next step Acquire the samples on the flow cytometer. For details,
go to bdbiosciences.com/cbasetup and select the
appropriate flow cytometer under CBA Kits: Instrument
Setup.
CBA samples must be acquired on the same day they are

prepared. Prolonged storage of samples, once the assay is
complete, can lead to increased background and reduced
sensitivity.
To facilitate the analysis of samples using FCAP Array

software, we recommend the following guidelines:
 Acquire standards from lowest (0 pg/mL) to highest

(Top Standard) concentration, followed by the test
samples.
 If running sample dilutions, acquire sequentially
starting with the most concentrated sample.
 Store all FCS files (standards and samples) in a single
folder.
When you are finished acquiring samples, proceed to
Data analysis (page 31).
Data analysis
How to analyze Analyze BD CBA Human Inflammatory Cytokines Kit
data using FCAP Array software. For instructions on
analysis, go to bdbiosciences.com/cbasetup see the Guide
to Analyzing Data from BD CBA Kits Using FCAP
Array Software.
Typical data The following data, acquired using BD CellQuest

software, shows standards and detectors alone.
Negative control: 0 pg/mL Standard: 20 pg/mL
Standard: 156 pg/mL Standard: 1,250 pg/mL
Standard curve The following graphs represent standard curves from the
examples BD CBA Human Inflammatory Cytokine Standards.
r Scale
cale
5
Performance
 Theoretical limit of detection (page 35)

 Recovery (page 36)
 Linearity (page 38)
 Specificity (page 40)
 Precision (page 41)
Theoretical limit of detection

Experiment The individual standard curve range for a given protein
details defines the minimum and maximum quantifiable levels
(for example, 20 pg/mL and 5,000 pg/mL) using the BD
CBA Human Inflammatory Cytokines Kit. By applying
the four-parameter curve fit option, it is possible to
extrapolate values for sample intensities not falling
within the limits of the standard curve. It is up to the
researcher to decide the best method for calculating
values for unknown samples using this assay. The
theoretical limit of detection for each protein using the
BD CBA Human Inflammatory Cytokines Kit is defined
as the corresponding concentration at two standard
deviations above the median fluorescence of
20 replicates of the negative control (0 pg/mL).
Limit of
detection data Limit of
Median Standard detection
Cytokine fluorescence deviation (pg/mL)
IL-8 3.4 0.4 3.6
IL-1 3.7 0.3 7.2
IL-6 4.7 0.4 2.5
IL-10 4.1 0.4 3.3
TNF 3.9 0.3 3.7
IL-12p70 4.0 0.3 1.9
Chapter 5: Performance 36
Recovery
Experiment Individual proteins were spiked into various matrices at
details three different levels within the linear assay range. The
matrices used in these experiments were not diluted
before addition of the protein. The plasma samples in
these experiments were EDTA treated. Results are
compared with the same concentrations of the proteins
spiked in the Assay Diluent.
Recovery data
Standard Observed in
spike conc. given matrix
Protein Matrix (pg/mL) (pg/mL) % Recovery
IL-8 Pooled donor 2,500 2,145 86%
sera (n=5) 625 548 88%
80 66 82%
Pooled donor 2,500 2,051 82%
plasmas (n=5) 625 473 76%
80 53 67%
Cell culture 2,500 2,709 108%
supernatant 625 661 106%
80 91 113%
IL-1 Pooled donor 2,500 1,656 66%
sera (n=5) 625 424 68%
80 42 52%
Pooled donor 2,500 1,776 71%
plasmas (n=5) 625 403 65%
80 47 58%
Cell culture 2,500 2,813 113%
80 81 101%
IL-6 Pooled donor 2,500 1,713 69%
sera (n=5) 625 484 78%
80 55 69%
Pooled donor 2,500 2,254 90%
plasmas (n=5) 625 573 92%
80 73 92%
Cell culture 2,500 2,646 106%
80 81 101%
IL-10 Pooled donor 2,500 2,016 81%
sera (n=5) 625 556 89%
80 67 84%
Pooled donor 2,500 2,308 92%
plasmas (n=5) 625 589 94%
80 70 88%
Cell culture 2,500 2,449 98%
80 79 99%
TNF Pooled donor 2,500 1,507 60%
sera (n=5) 625 419 67%
80 45 56%
Pooled donor 2,500 1,851 74%
plasmas (n=5) 625 457 73%
80 55 69%
Cell culture 2,500 2,864 115%
80 88 110%
IL-12p70 Pooled donor 2,500 1,366 55%
sera (n=5) 625 368 59%
80 46 57%
Pooled donor 2,500 1,752 70%
plasmas (n=5) 625 409 65%
80 53 66%
Cell culture 2,500 2,540 102%
80 81 101%
Linearity
Experiment In two experiments, the following matrices were spiked
details with IL-8, IL-1, IL-6, IL-10, TNF, and IL-12p70 and
were then serially diluted with Assay Diluent.
Linearity data
Observed
IL-8 IL-1 IL-6 IL-10 TNF IL-12p70
Matrix Dilution (pg/mL) (pg/mL) (pg/mL) (pg/mL) (pg/mL) (pg/mL)
Cell Neat 5,200 5,800 5,200 5,100 6,000 6,000
culture 1:2 2,520 2,665 2,590 2,494 2,597 2,560
media 1:4 1,260 1,291 1,280 1,302 1,241 1,283
1:8 602 596 635 643 606 618
1:16 296 312 310 329 320 302
1:32 154 143 150 156 157 154
1:64 74 74 75 74 78 72
1:128 39 39 41 40 45 42
1:256 19 17 20 19 23 20
Slope 1.01 1.04 1.01 1.01 0.99 1.02
Pooled Neat 4,468 2,884 3,410 4,662 3,213 2,962
human 1:2 2,583 1,815 2,016 2,452 2,038 1,757
sera 1:4 1,363 900 1,017 1,204 1,041 963
(n=5) 1:8 618 458 486 559 519 470
1:16 322 248 278 304 302 252
1:32 129 102 99 129 124 109
1:64 76 61 69 79 83 67
1:128 39 46 32 36 35 30
1:256 19 26 14 18 17 15
Slope 1.00 0.88 1.00 1.01 0.95 0.97
Pooled Neat 4,493 3,692 4,734 5,408 4,284 4,391
human 1:2 2,582 2,049 2,654 4,284 2,270 2,359
plasma 1:4 1,405 1,130 1,301 2,270 1,223 1,218
(n=5) 1:8 697 572 651 1,223 648 643
1:16 347 275 315 648 294 309
1:32 169 138 160 294 166 165
1:64 80 72 81 166 84 83
1:128 43 37 43 84 44 42
1:256 22 23 19 44 20 21
Slope 0.98 0.95 1.00 0.91 0.96 0.97
Specificity
Experiment The antibody pairs used in the BD CBA Human
details Inflammatory Cytokines Kit assay have been screened
for specific reactivity with their corresponding specific
proteins. Analysis of samples containing only a single
recombinant protein found no cross-reactivity or
background detection of protein in other Capture Bead
populations using this assay.
Specificity data Data for the detection of individual proteins was

analyzed using BD CellQuest software.
Human IL-8 Human IL-1 Human IL-6
Human IL-10 Human TNF Human IL-12p70
Precision
Intra-assay Ten replicates of each of three different levels of IL-8,
precision IL-1, IL-6, IL-10, TNF, and IL-12p70 were tested.
IL-8 80 pg/mL 625 pg/mL 2,500 pg/mL

Actual mean conc. 74 578 2,221
(pg/mL)
SD 3 12 104
%CV 4% 2% 5%
IL-1 80 pg/mL 625 pg/mL 2,500 pg/mL

(pg/mL)
SD 5 19 129
%CV 7% 4% 6%

(pg/mL)
SD 5 25 163
%CV 6% 5% 8%
IL-10 80 pg/mL 625 pg/mL 2,500 pg/mL

(pg/mL)
SD 5 25 121
%CV 6% 5% 6%
TNF 80 pg/mL 625 pg/mL 2,500 pg/mL

(pg/mL)
SD 6 34 207
%CV 9% 6% 10%
IL-12p70 80 pg/mL 625 pg/mL 2,500 pg/mL

(pg/mL)
SD 3 19 132
%CV 4% 3% 6%
Inter-assay Three different levels of IL-8, IL-1, IL-6, IL-10, TNF,

precision and IL-12p70 (80, 625, and 2,500 pg/mL) were tested in
four experiments conducted by different operators.
Note: The number of replicates refers to the total

number of assay tubes tested at a given concentration of
protein.

Number of 8 8 8
replicates
(pg/mL)
SD 3 24 172
%CV 4% 4% 7%
IL-1 80 pg/mL 625 pg/mL 2,500 pg/mL

Number of 8 8 8
replicates
(pg/mL)
SD 9 55 196
%CV 13% 9% 11%

Number of 8 8 8
replicates
(pg/mL)
SD 6 55 271
%CV 8% 9% 10%
IL-10 80 pg/mL 625 pg/mL 2,500 pg/mL

Number of 8 8 8
replicates
(pg/mL)
SD 6 57 306
%CV 8% 9% 11%
TNF 80 pg/mL 625 pg/mL 2,500 pg/mL

Number of 8 8 8
replicates
(pg/mL)
SD 6 94 351
%CV 8% 15% 13%
IL-12p70 80 pg/mL 625 pg/mL 2,500 pg/mL

Number of 8 8 8
replicates
Actual mean 77 654 2,563
conc. (pg/mL)
SD 5 49 223
%CV 6% 7% 9%
6
Reference
 Troubleshooting (page 45)

 References (page 47)
Troubleshooting
Recommended These are the actions we recommend taking if you
actions encounter the following problems.
For best performance, vortex samples immediately

before analyzing on a flow cytometer.
Problem Recommended action

Variation between Vortex the Capture Beads before pipetting. Beads can
duplicate samples aggregate.
Low bead number in  Avoid aspiration of beads during the wash step.
samples  Do not wash or resuspend beads in volumes
higher than the recommended volumes.
High background  Test various sample dilutions, the sample may be
too concentrated.
 Remove excess Human Inflammatory Cyokines
PE Detection Reagent by increasing the number
of wash steps, as the background may be due to
non-specific binding.
Little or no detection of Sample may be too dilute. Try various sample
protein in sample dilutions.
Less than six bead  Ensure that equal volumes of beads were added
populations are observed to each assay tube.
during analysis or  Vortex Capture Bead vials before taking aliquots.
distribution is unequal Once Capture Beads are mixed, vortex to ensure
that the beads are distributed evenly throughout
the solution.
Chapter 6: Reference 46
Problem Recommended action

Debris (FSC/SSC) during  Increase the FSC threshold or further dilute the
sample acquisition samples.
 Increase the number of wash steps, if necessary.
 Make a tighter FSC/SSC gate around the bead
population.
 Centrifuge or filter samples to reduce debris
before staining samples with the BD CBA Human
Inflammatory Cytokines Kit.
Overlap of bead This may occur in samples with very high protein
population fluorescence concentration. Ensure that instrument settings have
(FL3) during acquisition been optimized using the Cytometer Setup Beads.
Standards assay tubes  Verify that all components are properly prepared
show low fluorescence or and stored.
a poor standard curve  Use a new vial of standards with each
experiment, and once reconstituted, do not use
after 12 hours.
 Ensure that incubation times were appropriate.
All samples are positive or Dilute the samples further. The samples may be too
above the high standard concentrated.
mean fluorescence value
Biohazardous samples It is possible to treat samples briefly with 1%
paraformaldehyde before acquiring on the flow
cytometer. However, this may affect assay
performance and should be validated.
References
Related 1. Bishop JE, Davis KA. A flow cytometric
publications immunoassay for beta2-microglobulin in whole
blood. J Immunol Methods. 1997;210:79–87.
2. Camilla C, Defoort JP, Delaage M, et al. A new flow
cytometry-based multi-assay system. 1. Application
to cytokine immunoassays. Cytometry Suppl.
1998;8:132.
3. Carson R, Vignali D. Simultaneous quantitation of
15 cytokines using a multiplexed flow cytometric
assay. J Immunol Methods. 1999;227:41–52.
4. Chen R, Lowe L, Wilson JD, et al. Simultaneous
quantification of six human cytokines in a single
sample using microparticle-based flow cytometric
technology. Clin Chem. 1999;45:1693–1694.
5. Collins DP, Luebering BJ, Shaut DM. T-lymphocyte
functionality assessed by analysis of cytokine
receptor expression, intracellular cytokine
expression, and femtomolar detection of cytokine
secretion by quantitative flow cytometry. Cytometry.
1998;33:249–255.
6. Fulton RJ, McDade RL, Smith PL, Kienker LJ,
Kettman JR Jr. Advanced multiplexed analysis with
the FlowMetrix system. Clin Chem. 1997;43:1749–
1756.
7. Kricka LJ. Simultaneous multianalyte
immunoassays. Diamandis EP, Christopoulos TK,
eds. Immunoassay. Academic Press. 1996:389–404.
8. Lund-Johansen F, Davis K, Bishop J, de Waal
Malefyt R. Flow cytometric analysis of
immunoprecipitates: high-throughput analysis of
protein phosphorylation and protein-protein
interactions. Cytometry. 2000;39:250–259.
9. Fulwyler M, McHugh TM. Flow microsphere

immunoassay for the quantitative and simultaneous
detection of multiple soluble analytes. Methods Cell
Biol. 1994;33:613–629.
10. Oliver KG, Kettman JR, Fulton RJ. Multiplexed
analysis of human cytokines by use of the
FlowMetrix system. Clin Chem. 1998;44:2057–
2060.
11. Stall A, Sun Q, Varro R, et al. A single tube flow
cytometric multibead assay for isotyping mouse
monoclonal antibodies. Abstract LB77.
Experimental Biology Meeting 1998 (late-breaking
abstracts).
12. Cook EB, Stahl JL, Lowe L, et al. Simultaneous
measurement of six cytokines in a single sample of
human tears using microparticle-based flow
cytometry: allergics vs non-allergics. J Immunol
Methods. 2001;254:109–118.
13. Dotti G, Savoldo B, Takahashi S, et al. Adenovector-
induced expression of human-CD40-ligand
(hCD40L) by multiple myeloma cells: A model for
immunotherapy. Exp Hematol. 2001;29:952–961.
Notes
Notes
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Technical Service 877.232.8995
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Catalog No. 551811
23-11112-01 Rev. 03
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BD Cytometric Bead Array

(CBA) Human Inflammatory

Catalog No. 551811

Falcon® is a registered trademark of Corning Incorporated.

BD flow cytometers are Class 1 Laser Products.

Chapter 1: About this kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

 Purpose of the kit (page 6)

Purpose of the kit

Principle of CBA BD CBA assays provide a method of capturing a soluble

Each capture bead in the kit has been conjugated with a

When the capture beads and detector reagent are

Principle of this Six bead populations with distinct fluorescence

During the assay procedure, you will mix the

Advantages over The broad dynamic range of fluorescent detection via

 The required sample volume is approximately one-

The theoretical limit of detection of the BD CBA Human

The BD CBA Kit is not recommended for use on stream-

Serum spike recoveries for IL-1, TNF, and IL-12p70 are

The sensitivity for the detection of IL-1 in this assay is

Quantitative results or protein levels for the same sample

The BD CBA Human Inflammatory Cytokines Kit assay

Bead reagents Human Inflammatory Cytokine Capture Beads (A1–

Cytometer Setup Beads (D): A 30-test vial of setup beads

Antibody and Human Inflammatory Cytokine PE Detection Reagent

Human Inflammatory Cytokine Standards (C): Two

PE Positive Control Detector (E1): A 10-test vial of

FITC Positive Control Detector (E2): A 10-test vial of

Buffer reagents Wash Buffer (F): A 260-mL bottle of phosphate buffered

Assay Diluent (G): A 30-mL bottle of a buffered protein

Serum Enhancement Buffer (H): A 10-mL bottle of a

Note: Source of all serum proteins is from USDA-

Storage and handling

Appearance: The visual appearance of Assay Diluent

Warning Components A1–A6, B, D, E1–E2, F, G, and H contain

Human Inflammatory Standards (component 51-

Hazard May cause an allergic skin reaction.

Precautionary Wear protective gloves / eye protection.

Avoid breathing mist/vapours/spray.

If skin irritation or rash occurs: Get medical advice/

IF ON SKIN: Wash with plenty of water.

Dispose of contents/container in accordance with local/

 Workflow overview (page 15)

Procedure Incubation time

 A dual-laser flow cytometer equipped with a 488-

 Falcon® 12 × 75-mm sample acquisition tubes for a

Materials  Millipore MultiScreenHTS-BV 1.2 µm Clear non-

 Preparing Human Inflammatory Cytokines Standards (page 19)

Preparing Human Inflammatory Cytokines Standards

You must prepare fresh standards to run with each

Procedure To reconstitute and serially dilute the standards:

b. Continue making serial dilutions by transferring

6. Prepare one 12 × 75-mm tube containing only Assay

Concentration of See the Performing the Human Inflammatory Cytokine

Next step Proceed to Mixing Human Inflammatory Cytokine

Mixing Human Inflammatory Cytokine Capture Beads

Mixing the beads To mix the Capture Beads:

3. Add a 10-µL aliquot of each Capture Bead, for each

Resuspending If you are using serum or plasma samples, you must

To resuspend the Capture Beads in Serum Enhancement