The Mouse IL-6 Standard has been tested to assure to function as a standard when used with the BD™ Cytometric Bead Array (CBA). Investigators are encouraged to refer to the technical data sheet for the BD™ CBA Mouse IL-6 Flex Set (Cat. No. 558301) or the BD™ CBA Mouse IL-6 Enhanced Sensitivity Flex Set (Cat. No. 562236). The Mouse IL-6 Standard is lyophilized and should be reconstituted and serially diluted before mixing with CBA capture beads and PE detection reagents. An abbreviated instruction has been provided below (Preparation of Mouse IL-6 Standard Protocol). The Mouse IL-6 Standard should be reconstituted using Assay Diluent, which is provided with the BD™ CBA Mouse/Rat Soluble Protein Master Buffer Kit (Cat. No. 558266 or 558267), the BD™ CBA Mouse Enhanced Sensitivity Master Buffer Kit (Cat. No. 562246 or 562248) or it may also be purchased separately (Cat. No. 560104).
Preparation of Mouse IL-6 Standard for use with the BD™ CBA Mouse IL-6 Flex Set (Cat. No. 558301):
1. Open one vial of the lyophilized Mouse IL-6 Standard. Transfer the lyosphere(s) to a polypropylene tube (BD Falcon™, Cat. No. 352097). Label the tube "Top Standard".
2. Reconstitute the standard with 4.0 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 500 µl of Assay Diluent to each of the dilution tubes.
5. Perform a serial dilution by transferring 500 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 500 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.
Preparation of Mouse IL-6 Standard for use with the BD™ CBA Mouse IL-6 Enhanced Sensitivity Flex Set (Cat. No. 562236):
1. Open one vial of the lyophilized Mouse IL-6 Standard. Transfer the lyosphere(s) to a polypropylene tube (BD Falcon™, Cat. No. 352097).
2. Reconstitute the standard with 4.0 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, 1:3, 1:9, 1:27, 1:81, 1:243, and 1:729.
4. Add 460 µl of Assay Diluent into the Top Standard tube.
5. Add 400 µl of Assay Diluent to each of the remaining dilution tubes.
6. Transfer 40 µl of the reconstituted standard into the Top Standard tube. Mix by pipette or gently vortexing for no longer than 3 seconds.
7. Perform a serial dilution by transferring 200 µl from the Top Standard to the 1:3 dilution tube and mix thoroughly. Do not vortex, mix by pipet only. Continue making serial dilutions by transferring 200 µl from the 1:3 tube to the 1:9 tube and so on to the 1:729 tube and mix thoroughly. Prepare one tube containing only Assay Diluent to serve as the 0 fg/mL negative control.
