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CBA Mouse Inflammation 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 Inflammation Standards and the Cytometer
Setup controls in each experiment. See Table 2 for a detailed description of the
reagents added to the Inflammation Standard control assay tubes. The Cytometer
Setup procedure is described in Cytometer Setup, Data Acquisition, and Analysis
on pg. 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 Inflammation 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 Inflammation PE Detection Reagent to the assay
tubes.
5. Incubate the assay tubes for 2 hours at RT and protect from direct exposure
to light. During this incubation, perform the Cytometer Setup procedure
described in-
Cytometer Setup, Data Acquisition, and Analysis on pgs. 13 – 14.
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.
