Example 6
0.1×106 lymphocytes were incubated with a mix of Th1 cytokine (IFN-γ and IL-2) or Th2 cytokine (IL-4, IL-5, and IL-10) capture microparticles (8,000/each with different fluorescent ID codes) in control media or the conditioned media containing various types of immunotherapeutic agents (Solumedrol 100 μM, Pharmacia & Upjohn Co.; Sirolimus 500 ng/mL, Wyeth pharmaceuticals Inc.; Prograf 100 ng/mL, Astellas Pharma US, Inc.; Infliximab 1 mg/mL, Janssen Biotech, Inc.) at 37° C. in a 5% CO2 incubator for 16 h with and without adding PHA or P+I (50 ng/mL of PMA+1 μg/mL of lonomycin). The cells were lysed with lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40) and washed three times with 3% HBSA wash buffer (3% Bovine Serum Albumin in Hank's Balanced Salt Solution), the capture microparticles were then incubated with 50 μL detection antibodies mix (PE-anti-INF-γ, PE-anti-IL-2, PE-anti-IL-4, PE-anti-IL-5 and PE-anti-IL-10) from BD Bioscience at RT for 1 h with shaking. After three more washes, the microparticles were acquired and analyzed on a BD FACSCanto II flow cytometer. The result was expressed as a percentage of each of the different positive cytokine capture microparticles.