When T cells are stimulated by non-specific mitogens (e.g. PHA, ConA) during in vitro culture, they may be transformed into lymphoblastoid cells with increased cell size, vigorous metabolism, and increased synthesis of proteins and nucleic acids. (Source: Immunology Internship Guidance - Department of Immunology, School of Basic Medical Sciences, Peking University School of Medicine)
Operation method
T-lymphocyte transformation assay
Principle
When T cells are stimulated by non-specific mitogens (e.g. PHA, ConA) during in vitro culture, they can be transformed into lymphoblastoid cells by increasing cell size, metabolism, protein and nucleic acid synthesis. The level of lymphocyte transformation rate can reflect the level of cellular immunity of the body, so it can be used as one of the indicators to determine the body's immune function. There are three kinds of lymphocyte transformation tests: morphology counting method, MTT method and isotope method. MTT method is the colorimetric method of trace enzyme reaction of tetramethyl azole salt, MTT is a thiazole salt, chemically known as 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide, and the aqueous solution is yellow-orange in color. Mouse splenocyte proliferation activation after ConA action, its intracellular mitochondrial succinate dehydrogenase activity is correspondingly elevated, MTT as a substrate to participate in the reaction, the formation of blue dirty (Formazan) particles deposited in the cell or around the cell, the hydrochloric acid - isopropanol dissolved in blue solution, the OD value of the cell cultures can be measured with the enzyme labeling instrument, the measurement of the wavelength of 570 nm. According to the size of the OD value to calculate the degree of cell proliferation in the reaction system.3H-TdR doping method: the basic conditions or prerequisites for cell proliferation for the replication of the cytoplasm and nucleus, which is an indispensable prerequisite for the normal cell proliferation process. Generally speaking, a cell cycle can be roughly divided into four phases, i.e., G1 phase, S phase, G2 phase and M phase. The S phase is the DNA synthesis phase, and the main functional activity is DNA synthesis. 3H-TdR, or (methyl-3H)thymidine, is the precursor of DNA synthesis. It is added to cell cultures and taken up by cells as a raw material for DNA synthesis. The more DNA synthesized by the cell, the more 3H-TdR is incorporated, and therefore, the degree of cell proliferation can be reflected by detecting the incorporated 3H-TdR. Since this method has the problem of isotope contamination, we still use the MTT method in our internship.
Materials and Instruments
ICR Mouse Move 1. Preparation of mouse spleen cell suspensions Caveat 1. Since this experiment needs to be incubated for 3 days before the results can be observed. Therefore, attention should be paid to aseptic operation to avoid bacterial contamination, which may lead to the failure of the experiment. 2. Cell manipulation should be gentle and rapid, so as to avoid cell damage affecting the experimental results. Common Problems For more product details, please visit Aladdin Scientific website.
RPMI1640 culture medium Hank's solution Knife protein A MTT Iodine Alcohol
Sterile Tip Pipettes Graduated Pipettes Sterile Dissecting Instruments Flat-Bottom Culture Plates CO2 Incubator Enzyme Labeling Instruments
A sterilized flat dish was taken and 5 ml of Hank's solution was added. Mice were executed by cervical dislocation method, spleen was taken, placed in the flat dish, ground and sieved on a stencil to make cell suspension. 100 μl was removed for counting. Transfer the rest of the cell suspension into a centrifuge tube, centrifuge at 1500 rpm for 7 min (or 1000 rpm for 10 min), discard the supernatant, and dilute with RPMI1640 culture medium to make a 2. 5×106 /ml spleen cell suspension, then add ConA to make the final concentration of 2 μg/ml in each well, and make negative control wells without adding ConA at the same time. 2. Add the above cell suspension into 96-well flat-bottom culture plate, 0.1 ml per well.
3. Place the plate in a 37 ℃ incubator with 5 % CO2 for 48-72 hours, and 4-6 hours before the end of incubation, add 1 mg/ml MTT solution into each well of the plate, 10 μl/well. incubate at 37 ℃ for 6 hours. 4. 4. 110 μl of 0.01 M hydrochloric acid-isopropanol was added to each well and the OD value was measured using an enzyme labeling instrument at 570 nm within 30 min (or 100 ul/well of 2 % SDS overnight).
The OD values of the three replicate wells of the experimental and control groups were averaged.