The Human Inflammatory Cytokine Standard has been tested with the BD™ CBA Human Inflammatory Cytokine Kit (Cat. No. 551811) to assure protein amounts and function in the BD™ CBA assay. Investigators are encouraged to refer to the CBA kit manual or to the abbreviated instruction below (Preparation of Human Inflammatory Cytokine Standard Protocol). The Human Inflammatory Cytokine Standard should be reconstituted using Assay Diluent supplied with the aforementioned CBA kits (component no. 51-2432KC) or it may be purchased separately (Cat. No. 560104).
Preparation of Human Inflammatory Cytokine Standard : The Human Inflammatory Cytokine Standard is lyophilized and should be reconstituted and serially diluted before mixing with CBA capture beads and PE detection reagents.
1. Open one vial of the lyophilized Human Inflammatory Cytokine Standard. Transfer all the lyospheres to a polypropylene tube (BD Falcon™, Cat. No. 352097). Label the tube "Top Standard".
2. Reconstitute the standard with 2.0 mL of Assay Diluent. Allow the reconstituted standard to equilibrate for at least 15 minutes before making dilutions. Mix the reconstituted protein by pipette only. Do not vortex or mix vigorously.
3. Label additional 12 x 75 mm tubes (BD Falcon™, Cat. No. 352008) and arrange them in the following order: Top Standard, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, and 1:256.
4. Add 300 µl of Assay Diluent to each of the dilution tubes.
5. Perform a serial dilution by transferring 300 µl from the Top Standard to the 1:2 dilution tube and mix thoroughly. Do not vortex, mix by pipet only. 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 and mix thoroughly. Prepare one tube containing only Assay Diluent to serve as the 0 pg/mL negative control.