125I-UdR release assay to determine NK cell activity

Summary

Natural killer (NK) cells are involved in the first line of the body's immune defense and are mainly used in (1) anti-tumor and anti-virus (2) immunomodulation in the immune surveillance system.

Operation method

125I-UdR release assay to determine NK cell activity

Principle

25I-UdR (125I-2′ deoxyuridine nucleoside) is an analog of thymine nucleoside, which serves as a precursor for DNA synthesis, and can quite specifically replace thymine nucleoside doped into the DNA strand of the cell nucleus. Therefore, in vitro NK cell activity assays can be performed using 125I-UdR labeled in vitro passaged cultured tumor cells as target cells, and single nucleated cells isolated from peripheral blood or mouse splenocytes as effector cells. The lysis of target cells killed by effector cells releases125 I-UdR, and the radioactive intensity is measured by γ-counter, and the activity of NK cells is expressed as the percentage of 125I-UdR release.

Materials and Instruments

YAC-1 cells
125I-UdR (125I-2′ deoxyuridine nucleoside) Trypsin DNAase 5-fluorodeoxyuridine nucleoside RPMI-1640 culture medium
Plastic test tubes Gamma-counter Centrifuge

Move

I. MATERIALS

1. 125I-UdR : The physical half-life of 125I is 59.7d.

2. Trypsin : Prepared as 3mg/ml with Ca2+ and Mg2+ free Hanks solution, dispensed in small quantities and frozen at -20℃.

3. DNA enzyme: Prepared as 50μg/ml with Hanks liquid, small portions, frozen at -20℃.

4. 5-Fluorodeoxyuridine nucleoside (5-FudR) was prepared as 1×10-3 mol/L with saline and stored in 4℃ refrigerator.

5. The remaining materials were as above.

II. Methods of operation

1. Preparation of target cells: take 1ml (5×105 /ml) of target cells cultured for 24～48h, add 4～6μl of 5-FudR and 6μCi of 125I-UdR respectively, mix well, and incubate at 37℃ for 2h, take out the target cells and then wash them with 1640 nutrient solution containing 5% NCS for 3 times, centrifuge at 1500r/min for 2min each time, and then finally wash them with complete RPMI- 1640 culture solution. 1640 culture medium for suspension, and count the live cells. The labeling rate was detected by γ-counter, and generally up to 1~2cpm/cell was sufficient;

2. Preparation of effector cells: Isolate PBMC or mouse splenocytes by conventional methods, and finally suspend with complete RPMI-1640 culture medium and count;

3. effector-target cell action: adjust the concentration of effector cells and labeled target cells, add them into plastic test tubes respectively, the ratio of effector/target cells is 100:1. at the same time, set up natural release control tubes with only labeled target cells, set up 3 duplicate tubes for each specimen and the control tubes, replenish the volume of each tube to 1 ml with complete RPMI-1640 culture medium, mix, centrifuge at 1500 r/min for 2 min The tubes should be centrifuged at 1500r/min for 2min to promote the effect of target cells, and then incubated at 37℃ with 5% CO2 for 18h. 4.

4. Enzyme treatment: take out the effect/target cell culture, centrifuge at 1500r/min for 2min, discard the supernatant, add 0.1ml of trypsin and DNA enzyme in each tube, mix well, and then put it in a 37℃ water bath for 30min to promote the release of 125I-UdR from the damaged target cells, and then add 0.8ml of cold Hanks' solution in each tube to terminate the enzyme reaction. 1500r/min centrifugation for 2min, and then add 0.8ml of cold Hanks' solution to terminate the reaction. Min centrifugation for 2min, and 0.5ml of supernatant from each tube was aspirated into another plastic test tube.

5. Radioactivity measurement and calculation of results: Measure the cpm value of supernatant and cell fraction of each tube by γ-counter, and calculate the release rate of125I-UdR and NK cell activity according to the following formula, and take the average value of three tubes as the NK cell activity.

125 I-UdR release rate (%) = (0.5 ml supernatant cpm value × 2)/(0.5 ml supernatant cpm value + 0.5 ml cell suspension cpm value) × 100%

NK cell activity (%) = test tube125 I-UdR release rate - control tube125 I-UdR natural release rate.

Generally, the natural release rate of125 I-UdR is required to be <10%. When YAC-1 cells are used as target cells for detecting NK activity in mouse splenocytes, they may not be subjected to enzyme treatment prior to the assay.

Caveat

1. The labeling method requires a long incubation time, and treatment with enzymes can increase the sensitivity of the method.

2. The key to obtaining better results lies in the generative treatment of the target cells before labeling. The key to obtaining better results lies in the pre-labeling treatment of the target cells. The cells are kept in a state of optimal viability during the logarithmic growth phase to achieve the optimal labeling rate and to increase the resistance of the cells to the toxic effects of the radioisotopes, thus reducing the natural release.

3. because if contamination occurs the cultured cells have to be discarded and started again.

Common Problems

1. NK activity calculation:

Isotope release % (NK activity) = (experimental group cpm a natural release cpm) / (maximum release cpm a natural release cpm) x 100

Natural release % = (natural release cpm/maximum release cpm) x 100

The isotope release % listed in this result represents the NK activity. 2.

2. Sodium chromate (Na51cro4) and 125I-UdR (125I-UdR) are commonly used to label NK activity, and both methods have their own advantages and disadvantages.

51Cr advantages: sensitive, simple, short process, good reproducibility, the disadvantage of a short half-life, high natural release.

Advantages of 125I-UdR: long half-life, low natural release, suitable for longer incubation time. Disadvantages are lower sensitivity and long process.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/