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Table 1. Mouse Inflammation Standard concentrations after dilution
Protein
(pg/ml)
Mouse IL-6
Mouse IL-10
Mouse MCP-1
Mouse IFN-γ
Mouse TNF
Mouse IL-12p70
Top
Standard
5000
5000
5000
5000
5000
5000
1:2
Dilution
Tube
2500
2500
2500
2500
2500
2500
1:4
Dilution
Tube
1250
1250
1250
1250
1250
1250
1:8
Dilution
Tube
625
625
625
625
625
625
1:16
Dilution
Tube
312.5
312.5
312.5
312.5
312.5
312.5
1:32
Dilution
Tube
156
156
156
156
156
156
1:64
Dilution
Tube
80
80
80
80
80
80
1:128
Dilution
Tube
40
40
40
40
40
40
1:256
Dilution
Tube
20
20
20
20
20
20
Preparation of Mixed Mouse Inflammation
Capture Beads
The Capture Beads are bottled individually, and it is necessary to pool the bead
reagents (A1 – A6) immediately before mixing them together with the PE Detection
Reagent, standards, or samples.
1. Determine the number of assay tubes (including standards and controls)
that are required for the experiment (eg, 8 unknowns, 9 standard dilutions,
and 1 negative control = 18 assay tubes).
2. Vigorously vortex each Capture Bead suspension for a few seconds
before mixing.
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-6 Capture Beads × 18 assay tubes = 180 µl of IL-6 Capture Beads
required).
4. Vortex the Bead mixture thoroughly.
The mixed Capture Beads are now ready to be transferred to the assay tubes (50
µl of mixed Capture Beads/tube) as described in BD CBA Mouse Inflammation
Assay Procedure, pg. 12.
Note: Discard excess mixed Capture Beads. Do not store after mixing.
Preparation of Test Samples
The standard curve for each protein 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 standard
curve. For best results, samples that are known or assumed to contain high levels
of a given protein 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.
