Determination of the biological activity of the interleukin-2 kit - PhD thesis - Dissertation

The biological activity of the Interleukin-2 (IL-2) kit is measured using the Shanghai Jinma Biologist IL-2 ELISA kit, which is available for various species including rats and mice. We offer competitive pricing during the second quarter and provide free technical support and testing services to ensure a seamless experience for your experiments. **Purpose** 1. Understand the principle behind measuring IL-2 biological activity. 2. Learn how to perform and interpret the results of IL-2 activity assays. **Experimental Principle** IL-2 is a cytokine produced by T helper (Th) cells and plays a crucial role in lymphocyte proliferation and differentiation. The IL-2 activity assay relies on the ability of IL-2 to support the growth and survival of IL-2-dependent cells. As these cells proliferate, their energy metabolism increases, leading to higher levels of DNA synthesis. This metabolic activity can be indirectly measured as an indicator of cell proliferation. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is a yellow water-soluble compound that is reduced by living cells—especially those in active proliferation—to form a blue formazan crystal. The amount of formazan formed is proportional to the number of viable cells. After dissolving the crystals with SDS, the optical density (OD) at 570 nm is measured, reflecting both cellular metabolism and IL-2 activity. **Materials** - 1640 culture medium (complete), IL-2 standards, IL-2 samples, CTLL-2 cell line, MTT solution (5 mg/mL), 10% SDS - 96-well plates, multi-channel pipette, micro-sampler, CO₂ incubator, microplate reader **Procedure** 1. Prepare CTLL-2 cell suspension: Harvest actively growing cells, wash three times with 1640 medium, and resuspend at 2×10⁵ cells/mL. 2. Dilute the sample and standard: Prepare serial dilutions of IL-2 samples and standards using complete 1640 medium. 3. Load and incubate: Add 50 μL of each dilution and control wells into a 96-well plate, then add 50 μL of cell suspension. Incubate at 37°C with 5% CO₂ for 36 hours. 4. Add MTT: Introduce 20 μL of MTT solution per well and incubate for 6–8 hours. 5. Dissolve formazan: Add 100 μL of 10% SDS per well and incubate at 37°C to dissolve the crystals. 6. Measure OD: Read the absorbance at 570 nm using a microplate reader. Compare the sample OD values to the standard curve to determine IL-2 activity. **Results Calculation** Convert the OD values of each dilution into a probability unit based on the maximum OD value. The S-shaped curve is linearized, and regression equations are derived from the data points. Determine the dilution corresponding to 50% of maximum proliferation and calculate the IL-2 activity using the formula: **Formula:** x = (a × d) / D Where: - x = IL-2 activity of the sample (μ/mL) - a = IL-2 activity of the standard (μ/mL) - d = dilution factor of the sample at 50% proliferation - D = dilution factor of the standard at 50% proliferation **Precautions** 1. MTT should be used fresh and stored away from light. If precipitates are present, filter before use. 2. The biological assay is highly sensitive and specific, detecting only biologically active IL-2. Unlike immunoassays, it does not measure immunoreactive IL-2 proteins. Therefore, strict adherence to protocols is essential for accurate results.

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