IL-2 biological activity assay
IL-2 biological activity assay
Interleukin-2 (IL-2) is a cell proliferation factor secreted by activated helper T cells, which has the effect of promoting T cell proliferation and maintaining long-term T cell growth in vitro.CTLL-2 is an IL-2-dependent cell line can be used as a quantitative assay for IL-2 biological activity. In this experiment, MTT assay was used to determine the IL-2 biological activity units by measuring the proliferation of CTLL-2 cells. Detection of IL-2 biological activity provides indirect insight into the function of helper T cells.
Operation method
MTT analysis
Materials and Instruments
Human Peripheral Blood T Cells Move I. Induction of IL-2 secretion by human peripheral blood T cells Caveat 1. CTLL-2 cell viability should be greater than 95%. 2. Because the specimen contains IL-4, etc., which can affect the measurement of IL-2, it is best to use anti-IL-4 mAb adsorbent to remove IL-4. For more product details, please visit Aladdin Scientific website.
FCS-RPMI-1640 culture medium Glutamine HEPES Penicillin Streptomycin PBSA buffer
Centrifuge Spectrophotometer Shaker
1. Human PBMC isolation: PBMC are isolated by concentration gradient centrifugation of conventional lymphocyte fractionation solution, and the cell concentration is adjusted to 10% FCS-RPMI-1640 culture medium.
2. Sampling: Inoculate cell suspension in 24-well culture plate, 0.51 ml per well, add PHA 0.5 ml per well, incubate at 37 degrees, 5% CO2 for 48 hours.
3. Recovery of supernatant: Aspirate the cell culture supernatant, centrifuge at 12000 r/min for 15 minutes, collect the supernatant and freeze at -20 degrees.
II. IL-2 bioactivity assay
1. CTLL-2 cell preparation: CTLL-2 cells in logarithmic growth phase of 24~48 hours of passaging culture were taken and washed twice by centrifugation with culture medium, centrifuged at 1000 r/min for 5 minutes each time, and then the cell concentration was modulated to 1×105/ml with 10% FCS-RPMI-1640 culture medium.
2. Sample addition: Inoculate the cells into 96-well culture plate, 0.1 ml per well, at the same time, the sample to be tested and the standard will be doubly diluted, adding 0.1 ml per well, and each dilution will be set up in 3 duplicate wells. A control well (100 ul cells + 100 ul culture medium) was also set up. incubate at 37 degrees Celsius, 5% CO2 for 40 hours.
3. MTT admixture: gently aspirate 100 ul of supernatant, add 100 ul of MTT (1 mg/ml) and incubate for 2 hours at 37 degrees Celsius with 5% CO2.
4. Measurement: Centrifuge the plate at 2000 r/min for 5 minutes, discard the supernatant, add 100 ul DMSO to each well and act for 30 minutes, then measure the absorbance value (A) at 570 nm with an enzyme meter.
5. Calculation: Plot the standard curve using the concentration of the standard and the corresponding net A570 nm value ( A570 nm value of the experimental wells - negative control wells). According to the standard curve to find out the IL-2 level of the samples to be tested.