The Rat TNF Standard has been tested to assure to function as a standard when used with the BD™ Cytometric Bead Array (CBA), such as the BD™ CBA Rat TNF Flex Set (Cat. No. 558309). Investigators are encouraged to refer to the technical data sheet for the BD™ CBA Rat TNF Flex Set (Cat. No. 558309). An abbreviated instruction has been provided below (Preparation of Rat TNF Standard Protocol). The Rat TNF 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) or it may also be purchased separately (Cat. No. 560104).
Preparation of Rat TNF Standard: The Rat TNF 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 Rat TNF 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.