Protocol for Detection of IL-4 by B-Cell Proliferation Assay

The stimulation of B lymphocytes by suboptimal doses of anti-IgM antibodies is weak. When interleukin 4 (IL-4) is added, B lymphocytes show a significant increase in DNA synthesis in response to stimulation with sub-adapted doses of anti-IgM antibody, and the amount of ³H-TdR doping correlates with the amount of IL-4. In this experiment, IL-4 has been detected by stimulating B cell proliferation with a sub-adapted dose of IsM antibody.

• BALB/c or C57BL/6 mice are taken and splenic lymphocytes are isolated by conventional methods. Wash with Hanks' solution twice; then adjust the cell concentration to 1×10⁷-2×10⁷/mL with 10% NBS-IMDM culture medium.
• Place the above suspension of splenocytes in a 24-well culture plate, 1-1.5 mL per well, and incubate in a 37°C, 5% CO₂ incubator for 1 h; transfer the cell suspension to another culture well, and continue to incubate for 1 h, and then collect the non-adherent splenocytes.
• Adjust the cell concentration to 5×10⁷/mL, add anti-mouse T-cell serum or anti-Thy-1, 2McAb at 1:40 (V/V). Set at 4°C for 30 min, then add fresh guinea pig serum at 1:15 (V/V), mix well, and incubate for 1 h; centrifuge and discard the supernatant, and suspend the cells in a serum-free culture medium. At this time, T cells are dead and broken, through the lymphocyte layering solution, low-speed centrifugation to be removed.
• Centrifuge with lymphocyte layering solution at 500-800 r/min for 6-8 min, and purify the above cell suspension again. Wash 1-2 times with Hank's solution and adjust the cell concentration to 1×10⁶-2×10⁶/mL.
• Add the above B-cell suspension into a 96-well culture plate, add the IL-4 supernatant to be tested or different amounts of standards, the concentration of which is 0.5 U, 1 U, 2 U, 3 U, 4 U, 5 U, 6 U per milliliter, and set up 3 duplicate wells for each group.
• Add a sub-adaptive dose of anti-IgM antibody, generally the final concentration of 0.5% (V/V) for the sub-adaptive dose of anti-IgM, can also be measured before the experiment.
• The cells are routinely cultured for 48-72 h, and ³H-TdR is added 8-16 h before the end of the incubation period, and the cells are harvested to determine the cpm value. The cpm value of the standard is used to draw a standard curve, and the IL-4 activity is found from the standard curve.

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• IL-4 is a pleiotropic cytokine produced by activated T cells, mast cells, and basophils. IL-4 stimulates the activation and differentiation of B cells as well as other hematopoietic and non-hematopoietic cells. Creative Bioarray provides high-quality Human Interleukin 4, Recombinant Human Interleukin 4, Recombinant Mouse Interleukin 4, Recombinant Human Interleukin 4 (for Cell Culture), Recombinant Human Interleukin 4 (E. coli), and others.

• The purity of B cells has a direct impact on the results. The presence of T cells or dead T cells and their fragments in the B cell suspension can affect the results.
• When purifying B cell suspension, the operation time should be shortened as much as possible to reduce the unfavorable factors and ensure the cell viability, so that the experimental results are more stable and reproducible.
• The harvesting time of induced cell supernatant must be controlled at about 30 h. If the time is too long, the IL-4 content in the supernatant will be reduced.

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