Detection of immune function of red blood cells

1. Principle

It has been proved that human and various animal red blood cells, like white blood cells, also have important immune functions. It is based on the C3b receptor (C3bR) on the surface of red blood cells. The C3bR on the surface of red blood cells can play a variety of functions such as clearing immune complexes (IC), promoting phagocytosis, presenting antigens, and activating complement. Therefore, the established methods for detecting the immune function of red blood cells are mostly designed based on C3bR on the surface of red blood cells. The established methods mainly include standard erythrocyte immune adhesion test, erythrocyte SPA mixed garland test, monoclonal antibody Coombs test, anti-C3bR monoclonal antibody method, enzyme-linked immunosorbent assay, radioligand binding method, erythrocyte phagocytosis determination Law etc.

This experiment only introduces the simple and easy erythrocyte C3bR yeast wreath test and erythrocyte IC yeast wreath test. The former is used to measure C3bR of red blood cells. The principle is that yeast sensitized with serum (complement) such as mice has C3b adsorbed on the cell surface, which can be combined with C3bR on the surface of red blood cells, thereby adsorbing yeast cells around the red blood cells to form a wreath. The latter is used to detect immune complexes on the surface of red blood cells. The principle is that C3bR on the surface of red blood cells can bind to the complement components of the antigen-antibody-complement complex formed in the body, and the immune complex is adsorbed on its surface, while yeast cells It can also be combined with complement components in immune complexes, thereby being adsorbed on the surface of red blood cells to form garlands. By detecting the level of the two types of garlands, the immune function status of red blood cells can be determined.

2. Materials and methods

(1) The materials mainly include centrifuges, constant temperature water baths, microscopes, counters, small centrifuge tubes, blood cell counting plates, yeast reagents, Hanks solution, guinea pig serum, animal anticoagulant, Ji's staining solution, etc.

(2) Method

1 Red blood cell C3bR yeast garland test

(1) Preparation of C3b-sensitized yeast suspension: Wash the yeast reagent with Hanks solution (containing calcium and magnesium, pH value 72, the same below) once (1000 r / min 10min) to prepare 1 × 108 yeast cells ml suspension, then add an equal amount of fresh guinea pig serum, mix well, 37 ° C water bath for 20min, and then washed twice with Hanks solution to restore the original concentration, which is C3b-sensitized yeast cell suspension. After the yeast reagent is washed with Hanks solution, it is prepared into a concentration of 1 × 108 / ml, which is a non-sensitized yeast suspension.

(2) Preparation of red blood cell suspension: human or rat, rabbit, cow, chicken and other tested animals heparin anticoagulated, washed 3 times with Hanks solution

(Centrifuged at 2000 r / min for 5 min each time), and finally the red blood cells were diluted with Hanks solution into a suspension of 125 × 107 cells / ml.

(3) Detection method: take 50μl each of C3b sensitized yeast suspension and erythrocyte suspension, mix well, take out after 40min water bath at 37 ° C, shake gently, add 20μl of 025% glutaraldehyde solution and fix for 5min, use appropriate amount of Hanks Dilute the solution, smear, dry, fix with methanol, stain with Giemsa's stain, high magnification examination, take 1 red cell adhered to 2 or more yeast cells as a garland, count 200 red cells, and find the garland rate.

2 Red blood cell IC yeast garland test Except that yeast cells are not sensitized, the rest are the same as red blood cell C3bR garland test.

3. Results

Under normal circumstances, both experiments will see a wreath formed by adhering several yeast cells around the red blood cells, and the red cell C3bR rosette rate is much higher than the red cell IC yeast rosette rate. Under normal circumstances, the rate of two types of garlands in humans is 25% -35% and 3-8%, respectively, and the rate of two types of garlands in chickens is 9% -19% and 3% -6%. But when a disease occurs (such as various infectious diseases and tumors), the two garland rates will change.

4. Matters needing attention

1 Complements of different animal sources have a great influence on the results of this experiment, so the detection of red blood cells of different species should use the complements of different animals. For the detection of human red blood cells, guinea pig serum should be used as complement, the detection of chicken red blood cells should use rabbit serum as complement, and the detection of bovine red blood cells should use mouse serum as complement. The source of complement is wrong and it is difficult to form a wreath.

2 Both the complement serum and the red blood cells to be tested should be collected and used. If they cannot be used immediately, they can be temporarily stored in a refrigerator at 4 ° C, but generally no more than 6h.

3 When the test tube is taken out of the water bath and shake well, it should be shaken gently with wrist force, and should not be shaken. When smearing, use a capillary to suck a small drop on the slide, and gently spread it with a flat capillary. Do not apply it repeatedly.

4 The pH value of the Hanks solution should be adjusted accurately, otherwise it is not easy to form a wreath.

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