Lymphocyte transformation assays are used in clinical practice to (1) reflect the response function of the overall level of T-cells in the subject, to determine the state of cellular immunity and to explore the pathogenesis (2) to select suitable transplant donors (3) to search for the cause of delayed-type metaplasia (4) to estimate the efficacy and prognosis of the disease.
Operation method
Lymphocyte transformation assay
Principle
T-lymphocytes from normal organisms are stimulated by specific antigens or mitogens during in vitro culture. Can be transformed into lymphoblastoid cells. In cellular immunodeficiency, with malignant tumors or certain other diseases. The rate of transformation is apparently reduced. The basic principle is to use T-lymphocyte-sensitive stimulants to stimulate T-lymphocytes in vitro, T-lymphocytes can undergo morphological and biochemical changes after stimulation, and some of the small lymphocytes are transformed into immature mother cells and undergo mitosis. The degree of T-cell growth is measured morphologically or by radionuclide, and the corresponding cellular function is assessed.
Materials and Instruments
Heparin Move 1. Take 1~2 ml of heparin anticoagulated blood (20 units of heparin in 1 ml of blood) and mix thoroughly. leave it at 37℃ for 1~2 hours. After erythrocyte precipitation, aspirate the plasma layer and piggyback some erythrocytes. Transfer to another sterile test tube. Count the number of white blood cells and centrifuge at 2000 rpm for 10 minutes. Discard the supernatant. Prepare a 1x107/mL cell suspension from the precipitated leukocytes using 199 culture medium. Caveat 1. The composition of the medium has a great influence on the conversion rate, pay attention to its expiration date. 2. calf serum needs to be inactivated before use. 3. Enough gas should be guaranteed to ensure sterility during culture. 4. The PHA dose is too large and toxic to the cells, the PHA transformation reaction dose is generally 10ml, the total amount of liquid in the culture flask should not be more than 2ml, it is too small to be enough for secondary lymphocyte transformation, it should be measured before the experiment. Common Problems Transformation criteria of lymphocytes 1. Calculate the transformation rate of lymphocytes: look at the head, body and tail segments of each slide under an oil microscope. One vertical column for each segment (the purpose is to reduce the error of uneven distribution of cells in the push slides) Formula: Lymphocyte transformation rate = number of transformed lymphocytes / (number of transformed lymphocytes + number of untransformed lymphocytes) × 100%. 2. 200-400 cells are counted on each slide, and the conversion rate is calculated as 50-100 cells in the head, 50-100 cells in the body, and 100-200 cells in the tail. It is generally recognized that the normal value of conversion rate is 60-70%. 3; 3. Currently, phytohemagglutinin (PHA) is most commonly used as a dividing agent to detect the conversion rate of lymphocytes in subjects to determine their cellular immune function, known as the PHA-stimulated lymphocyte conversion assay. For more product details, please visit Aladdin Scientific website.
199 culture fluid calf serum streptomycin penicillin PHA
Syringes Needles Test tubes Straws Centrifuges
2. Take 0.6 ml of the cell suspension ( 6x106 cells) and add it to a culture flask containing 2.4 ml of 199 culture medium.
3. Add PHA at 30 ug PHA per ml of nutrient solution.
4. Incubate at 37°C for 68-72 hours. During incubation, turn the culture flask 1~2 times a day.
5. After incubation. Shake the culture flask. Resuspend the cells and transfer into test tubes.
6. Centrifuge at 1000 rpm for 5 minutes.
7. Discard as much of the supernatant as possible, mix the precipitate, place a drop on a slide, push it into a blood film (head, body, and tail distinct) dry it, and stain it with Ritter's stain) for microscopic examination.
8. Calculate the conversion rate of lymphocytes: observe the head, body and tail segments of each slide under oil microscope. One vertical column for each segment (the purpose is to reduce the error of uneven distribution of cells in the push slides).
9. Count 200~400 cells per slide and calculate the conversion rate, including 50~100 cells in the head, 50~100 cells in the body and 100~200 cells in the tail. The normal value for the conversion rate is considered to be 60-70%.
