3H-TdR incorporation assay to detect IL-2 biological activity

【Materials and reagents】

(1) Reactive cells: CTLL-2, survival rate should be> 95%.

(2) IL-2 standard product: multiple dilutions to different concentrations.

(3) Sample to be tested: multiple dilutions to different concentrations.

(4) 10% Fcs-RPMI-1640 complete culture solution

(5) 3H-TdR

(6) 96-well culture plate, graduated pipette, capillary pipette, graduated centrifuge tube, centrifuge, sample adder (head), cell counting plate, inverted microscope, ultra-clean workbench, CO2 incubator, micro cell collector, glass Fiber filter paper, ß liquid scintillation counter.

【Steps】

(1) Wash CTLL-2 cells twice with 10% FCS-RPMI-1640 culture solution and centrifuge at 1000r / min for 5min to remove the original growth medium (containing IL-2)

(2) Adjust the cell concentration to 1 × 105 / ml with 10% Fcs-RPMI-1640 medium;

(3) Add IL-2 standards and samples to be tested at different dilution ratios to 96-well cell culture plates, 100 μL / well, each with 3 replicate wells, and culture medium controls;

(4) Add CTLL-2 cell suspension to each well, 100μl / well;

(5) Incubate at 37 ℃, 5% CO2 incubator for 24h;

(6) Add 3H-TdR to each well, 0.5μci / well, and continue to cultivate for 4-6h;

(7) Collect the cells on the glass fiber paper with a micro cell collector, and measure the cpm value of each well with a ß liquid scintillation counter. The best time to determine the CPM value is when all or most of the cells in the culture control (without IL-2) die and cells with IL-2 wells grow vigorously.

【Result analysis】

(1) The cpm value of each well is the average of multiple wells, and then subtract the culture control, which is the final c of each well.

(2) Calculate IL-2 activity unit (see IL-1, IL-2 MTT colorimetric detection method) can be calculated as follows:

Sample IL-2 activity unit

(U / ml)

=

Sample dilution up to 50% maximum proliferation 3H-TdR infiltration value

×

Standard product IL-2 activity unit

(U / ml)

Standard dilution up to 50% maximum proliferation 3H-TdR infiltration value

【Precautions】

(1) Use 125I-UdR instead of 3H-TdR for IL-2 activity detection. The advantage is that it does not require reagents and equipment required for ß liquid scintillation determination, only a small γ counter is needed, and it can save time and reduce errors in ß liquid scintillation operation. The disadvantage is that the 125I-UdR has a short half-life and requires regular supply. The basic method is the same as the 3H-TdR incorporation method, except that 125I-UdR is used instead of 3H-TdR, 0.2μci / well, and then 1mmol / L 5-fluorouracil 5μl is added, and the culture is continued for 24h, and the cells are collected to determine the cpm value using a gamma counter .

(2) For the remaining precautions, please refer to the MTT method for detecting IL-2 activity.