T cells in normal human peripheral blood bind directly to sheep red blood cells (SRBC) in vitro to form rosette clusters. This is because human T cell membranes have receptors, called E receptors, that bind to glycoproteins on the SRBC membrane. (Source: Immunology Internship Guide - Department of Immunology, College of Basic Medical Sciences, Peking University)
Operation method
E Rosette formation test
Principle
T cells in normal human peripheral blood bind directly to sheep red blood cells (SRBC) in vitro to form rosette clusters. This is because human T cells have receptors on their membranes that bind to glycoproteins on the SRBC membrane, called E receptors. It has been proved that the E receptor is a unique surface marker of human T cells, so this test can be used as the identification and counting of human peripheral blood T cells, and also as an indicator of the state of human cellular immunity, and one of the commonly used methods to isolate T cells. This experiment is a demonstration, students only see the results.
Materials and Instruments
Heparin Anticoagulated Human Peripheral Blood Move 1. take 1 ml of heparin anticoagulated blood and add 1 ml of Hank's solution, mix well and add it to 2 ml of layered solution with a sharp pipette. Caveat 1. SRBC should be used within 2 weeks after removal from the sheep. Common Problems I. Experimental results For more product details, please visit Aladdin Scientific website.
Lymphocyte stratification solution SRBC suspension Hank's solution NBS
Hematocrit plate Tip pipette Slide Rachel's Stain
2. Centrifuge the blood at 2000 rpm for 30 minutes and pipet the lymphocyte-rich suspension at the interface of the plasma and the layered solution into another clean test tube. The lymphocyte-rich suspension at the interface between the plasma and the stratified fluid is aspirated with a sharp pipette and placed in another clean tube. 3.
3. Centrifuge the cells twice at 1000 rpm for 10 minutes with Hank's solution to rinse the cells, and then add 1 ml of Hank's solution to the final supernatant. After discarding the supernatant, add 1 ml of Hank's solution, mix well, and add 0.02 ml to 0.38 ml of leukocyte counting solution with a microfuge, and then add the supernatant to the leukocyte counting solution on a blood plate under low magnification. The number of cells per milliliter was counted under low magnification on a blood counting plate and calculated according to the following formula. Then 107/ml cell suspension was prepared with Hank's solution. 4.
4. Take 0.1 ml of 107/ml cell suspension and add 0.1 ml of inactivated NBS absorbed by SRBC, then add 0.2 ml of 0.5 % SRBC suspension, mix well and put it in 37 ℃ for 5 minutes, then centrifuge it at 500 rpm for 5 minutes, and put it in the refrigerator at 4℃ for 2 hours or overnight.
5. Remove the tube, gently suspend the precipitated cells, take out a drop with a pointed pipette and place it on a slide, cover with a coverslip that has been filled with Rachel's stain, and observe the counting under high magnification microscope.