Human T and B lymphocytes have different receptors on their surface. Since human T lymphocytes have receptors on their surface that bind to sheep erythrocytes and can bind non-specifically to sheep erythrocytes to form E wreaths, T cells also become erythrocyte petal-forming cells.

Operation method

Measurement of T-lymphocytes in guinea pigs

Principle

According to the rate of E-rosette formation, the cellular immunity of the body can be indirectly determined. At present, it has been widely used in clinical practice as an indicator for determining the immune status of the population.

Materials and Instruments

Guinea Pig
Heparin Rabbit erythrocyte suspension Hanks solution Fetal bovine serum
Centrifuge Tubes Centrifuge Shaker

Move

1. Lymphocyte Extraction
(1) Take 2 ml of guinea pig anticoagulated blood and dilute it with 3 ml of Hanks solution. Add to 3 ml of lymphocyte layering solution above the liquid level (add gently along the wall of the tube, do not mix the two liquids).
(2) Centrifuge at 2000 rpm for 30 minutes, aspirate the lymphocyte layer into another tube and wash with 5 times the volume of Hanks solution for 1~2 times, (centrifuge at 1500 rpm for 10 minutes) and discard the supernatant. Figure 1 Blood cell layer after centrifugation with layering solution
2. Add 0.1 ml of absorbed fetal bovine serum and 0.2 ml of 0.5% rabbit erythrocyte suspension to the lymphocytes. Mix and water bath at 37°C for 5 minutes.
3. Centrifuge at 500 rpm for 5 minutes, place in a refrigerator at 4°C for 2 hours and discard most of the supernatant.
4. staining and observationGently suspend the deposited cells. Add a drop of Meilan solution to the suspension, mix, and after 10 minutes take a drop and place it on a slide and microscopically examine it with a coverslip.Count 200 lymphocytes under high magnification or oil microscope, any combination of 3 or more rabbit erythrocytes was considered positive and the percentage of wreath forming cells was calculated.

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