Cell proliferation assay: H-thymidine (3H-TdR) infiltration

Experimental principle:

T cells and B cells have receptors for recognition of antigens and mitogen receptors on the surface, and the corresponding lymphocyte clones can be proliferated under the stimulation of specific antigens. Phytohemagglutinin (PHA), concanavalin A (ConA), anti-CD2 and anti-CD3 McAb as a polyclonal stimulator can selectively stimulate T cell proliferation; while anti-IgM, staphylococcal A-containing bacteria (SAC) Lipopolysaccharide (LPS, which acts on mice) stimulates the proliferation of B cells; P. americana (PWM) and tumor stimulator PMA have a stimulating effect on the proliferation of T and B cells. It has recently been discovered that certain receptors in the VLA group of the integrin family can also activate T cells after binding to the corresponding ligand. The proliferation level of T cells was determined according to morphological or tritiated thymidine (3H-TdR) incorporation rate.

First, materials and reagents

1. Medium: RPMI-1640 or TC199 medium

2, PHA; same morphology method

3, calf serum

4, 3H-TdR; use a product with a specific activity of 2Mci ~ 10Mci / mg molecules, 1Mci / ml solution was diluted to 100uci / ml with physiological saline, and stored at 4 ° C for use. When it is used, it is diluted with a culture solution to a 10 uci/ml solution.

5, 3% glacial acetic acid

6, concentrated formaldehyde

7, 30% H2O2 solution

8, scintillation fluid

PPO (2,5 diphenyl oxazole); 5.00g

POPOP [1,4-bis(5-phenyloxazolyl-2benzene; 0.30g)

Anhydrous ethanol; 200.00ml

Toluene; 800.00ml

Mix

Second, the operation method

1. The culture medium was added with a double antibody (containing penicillin 10,000 U/ml, streptomycin 0.01 g/ml) and heparin solution (containing heparin 100 U/ml) in a volume of 1%. The pH was adjusted to 7.2-7.4 with 3.5% NaHCO3 solution, and then the inactivated calf serum was added to a concentration of 20%. Add PHA (50U ~ 75U / ml), aseptically dispensed into the culture bottle, 1ml per bottle.

2, aseptic operation blood collection, and anti-coagulation with heparin, while white blood cell count and classification count.

3. Take 0.1 ml of anticoagulated blood and add it to a culture flask containing 1 ml of culture solution, and connect 2 to 3 culture flasks for each blood sample. If the whole blood is replaced by plasma or isolated lymphocytes, it is better to add it to the culture flask.

4. Incubate at 37 ° C for 72 h, and add 3H-TdR1uci to each bottle from 16 h to 24 h before the end of the culture.

5. After the end of the culture, the culture was transferred to a centrifuge tube, and the culture flask was rinsed twice with 3% glacial acetic acid 6 ml, and the rinse solution was also placed in a centrifuge bottle, centrifuged at 2 000 r/min for 10 min, and the supernatant was removed. The precipitate was washed once more with 3% glacial acetic acid and centrifuged to remove the supernatant.

6. Add 30 ml of H2O2 solution to the precipitate, and leave it at 85 °C for 15 min. When the solution is colorless, add 0.5 ml of formic acid and continue heating (85 °C) for 30 min to completely digest and dissolve the precipitate.

7. Transfer the digested and dissolved liquid into the sample cup, bake it with infrared light or hot air at 80 °C until the liquid volume is less than 0.1 ml, add 5 ml of scintillation fluid, and measure the pulse number with a liquid scintillation counter.

8. Preparation of scintillation fluid samples in steps 5-7 can also be prepared by filter membrane method. Namely: after the completion of the culture, the culture solution was transferred to a centrifuge tube, centrifuged at 2000 r/min for 10 min, the supernatant was discarded, and the precipitate was washed once with physiological saline and centrifuged again. The precipitate was transferred to the filter (note that the precipitate was all transferred), filtered under reduced pressure, and sequentially with 10 ml of physiological saline, 5 ml of 5% trichloroacetic acid (if there is a colored substance, decolorized with 30% H 2 O 2 liquid) and 3 ml of no Water ethanol suction filtration. The filter was taken out and dried at 60 ° C to 80 ° C. Then, the filter was placed in a 5 ml scintillation fluid (faced with the cells facing up) and detected with a liquid scintillation counter.

Third, the result is judged

The number of pulses per minute was measured by a liquid scintillation counter. Take the average of the measured readings of each tube, which is the number of pulses of 0.1 ml blood sample. The number of pulses detected by whole blood was corrected to the number of pulses per million lymphocytes (cpm/106 lymphocytes) according to the results of blood sample lymphocyte counts. The stimulation index (SI) can also be used to indicate the test results. For this purpose, the same number of control flasks without PHA should be set during the culture. The ratio of the number of test tube pulses to the number of control tube pulses is the stimulation index.

Fourth, matters needing attention

1. The ratio of blood sample to medium is generally 1:10~1:30.

2. When culturing the cells, they can be cultured in a CO2 incubator or cultured in a common incubator, but the cap must be tightened, otherwise the results are quite different.

3. When the results are expressed in SI, be sure to set the same number of culture tubes (ie, no PHA). In the absolute value of cpm (cpm/106 lymphocytes), there is no need to set up a control tube. Because the control cpm is close to the background, it is not easy to determine the significance of the difference between the different samples.

4. The volume of the scintillation fluid is very low, so it must be dried before adding the scintillation fluid. However, when a certain amount of alcohol (such as anhydrous ethanol) is added, a small amount of water remaining after the treatment can be accommodated, but it is still required to be dried to a degree close to dryness, otherwise turbidity will occur and measurement cannot be performed. According to reports, if 1/3 of isooctylphenoxypolyethoxyethanol (TritonX-100) is added to the scintillation fluid, the water capacity can reach 15%. After digesting and dissolving the sample, the scintillation liquid can be added without drying. Determination.

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