The human Th1/Th2 cytokine standards are lyophilized and should be reconstituted and serially diluted before mixing with the capture beads and the PE-detection reagent.
1. Reconstitute one vial of lyophilized human Th1/Th2 cytokine standards with 0.2 mL of assay diluent to prepare a 10X bulk standard. Allow the reconstituted standard to equilibrate for at least 15 min before making dilutions. Agitate vial to mix thoroughly. Do not vortex.
2. Label 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.
3. Add 900 ¡L of assay diluent to the top standard tube.
4. Add 300 ¡L of assay diluent to each of the remaining tubes.
5. Transfer 100 ¡L of 10X bulk standard to the top standard tube and mix thoroughly.
6. Perform a serial dilution by transferring 300 ¡L from the top standard to the 1:2 dilution tube and mix thoroughly. Continue making serial dilutions by transferring 300 ¡L from the 1:2 tube to the 1:4 tube and so on to the 1:256 tube and mix thoroughly. The assay diluent serves as the negative control.
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