Hemolytic vacuole formation test
Hemolytic vacuole formation test
The hemolytic vacuole formation test is an in vitro method for the detection of individual antibody-forming cells (plasma cells), and is therefore also known as the in vitro antibody-forming cell (PFC) assay. This technique not only serves as a powerful tool for the study of basic immunological theories, but is also widely used for the detection of antibody-forming cells that produce IgM types (including other types of immunoglobulins and their subclasses). (Source: Laboratory Guidelines for Basic Medical Immunology, edited by Jin Boquan and Li Enshan, 1st edition, Beijing: World Book Publishing House, 1990).
Operation method
Hemolytic vacuole formation test
Principle
The basic principle of the hemolytic vacuole formation test is to mix the spleen cells of mice immunized with sheep erythrocytes with a certain amount of sheep erythrocytes, and with the participation of complement, the sheep erythrocytes around the antibody-forming cells that are sensitized by the antibody molecules are lysed, resulting in the formation of hemolytic vacuoles that can be seen with the naked eye. Depending on the method of operation, it can be divided into direct hemolytic vacuole test, indirect hemolytic vacuole test, agar solid-phase method, chamber liquid-phase method, single-cell-layer method and so on.
Materials and Instruments
Serum Move 1. Preparation of bottom agar dishes Caveat 1. 0.7% agar must be held in a water bath at 47-49 °C in a molten state. If the temperature is too high it will cause hemolysis of SRBC or the addedThe temperature is too high, which may result in hemolysis of SRBC or death of the added splenocytes. If the temperature is too low, the agar will solidify during the procedure and affect the preparation of the upper agar plate.Preparation2. Isolated splenocytes should be stored in a refrigerator at 4 ℃ to prevent antibody secretion and cell death. 3.3. All glassware and reagents should be pre-warmed when preparing the plates.All reagents should be pre-warmed and mixed with 0.7% agar quickly and thoroughly after being added to a Watt's tube.The reagents should be mixed with 0.7% agar quickly and thoroughly, then poured onto the bottom layer of agar immediately, avoiding air bubbles.4. The added complement should cover the surface agar evenly. 5.5. When preparing the bottom dish and the test dish, the dish must be placed on a level table to ensure that the agar surface is spread evenly.The agar surface should be flat. For more product details, please visit Aladdin Scientific website.
PBS SRBC RPMI1640
Petri dishes Flasks Water baths Fahrenheit tubes Syringes Sieves Penicillin vials Centrifuge tubes Samplers
The 1.4% agar was melted by heating and poured into petri dishes, 6 ml per dish. After solidification, put it in a wet box at 37 ℃ for reserve.
2. 0.7% agar melted by heating and added to the Watson's tube, each tube 2 ml, 47 ~ 49 ℃ water bath insulation spare.
3. Immunized mouse spleen cell suspension preparation
The mice were immunized intraperitoneally with 4×103 sheep erythrocyte (SRBC) saline suspension, and the control was an equal number of sheep erythrocyte (SRBC). The mice were immunized with 4×103 sheep erythrocytes (SRBC) in saline suspension, and the control was equal amount of saline. RPMI164。洗一次后置平皿中不锈钢筛网上，用注射器针芯研磨成悬液，洗涤两次，调整 The cell number was adjusted to 3~8×106/ml, and the number of live cells should be more than 90% by Taipan orchid staining. Place in the refrigerator at 4 ℃ for spare.
4. Preparation of test dishes
Sequentially, 0.1 ml each of 25% SRBC, shank-SRBC absorbed inactivated fetal bovine serum and spleen cell suspension preserved at 4 ℃ were added to 0.7% SRBC at 47-49 ℃. 0.1 ml each of 25% SRBC, shank-SRBC-absorbed inactivated fetal serum, and splenocyte suspension preserved at 4 ℃ were added to a preheated 47-49 ℃ Fahrenheit tube containing 0.7% agar, quickly mixed, and immediately poured into a bottom-lined dish. Add 0.1 ml each of fetal bovine serum and spleen cell suspension stored at 4 ℃ into a preheated 47～49 ℃ Watson's tube containing 0.7% agar, mix rapidly, pour immediately into a flat dish with bottom layer of agar, and incubate at 37 ℃ for 1 hour after solidification.
5. Add 0.5~1.0 ml of fresh guinea pig serum diluted 1:5~1:10 to cover the surface evenly, and incubate at 37 ℃ again for 30 minutes. incubate at 37 ℃ for 30 minutes, and then count the empty spots with the naked eye or with the aid of a magnifying glass.