ExerciseⅥ Erythrocyte Roset te Forming Cell Assay
Principle
The surface of peripheral T lymphocytes contains the receptor CD2，which binds to sheep erythrocytes（E receptor）.CD2 is not present on the surface of B lymphocytes.Therefore，when mixed with sheep erythrocytes in vitro，only T lymphocytes can form E rosettes with sheep erythrocytes using E receptors.This test can be used to detect the number and ratio of T lymphocytes in peripheral blood.
Materials
1.Ficoll-Hypaque（lymphocyte separation medium）：specific gravity 1.077±0.001 g/L.
2.Heparin solution（25 U/ml）.
3.Hank's balanced salt solution（without Ca2+，Mg2+），p H 7.2～7.6.
4.Alsever preservation solution.
5.Sheep erythrocytes：Wash fresh sheep blood preserved in the Alsever preservation solution and adjust the sheep erythrocyte suspension to 1%.
6.Absorbed and inactivated Fetal Calf Serum：Fetal Calf Serum is inactivated by incubation in 56℃water for 30 min.Then add the washed precipitated sheep erythrocytes at a ratio of 0.1ml per ml Fetal Calf Serum.Mix well.Put it in a 37℃incubator for 30 min.Centrifuge at 2000 rpm/min for 20 min.Remove the supernatant and preserve it at 4℃.
7.0.8%glutaraldehyde water solution.
8.0.2%cold methylene blue.
9.Syringes，test tubes，pipettes，horizontal centrifuge.
Methods
1.Draw 3ml of venous blood and add it to a test tube containing heparin.Then mix with an equal volume of Hank's solution.Add 3ml of diluted blood to another test tube containing 2ml lymphocyte separation medium.Carefully add the diluted blood so that the surface of the separation media is not disturbed.
2.Centrifuge in a horizontal centrifuge for 20 minutes at 2000 rpm.Remove the test tube carefully.
3.Carefully draw the white mononuclear cells（containing the lymphocytes）that are in the interface of the lymphocyte separation medium.Wash the cells with Hank's solution and adjust the cell concentration to 2×106/ml.(https://www.daowen.com)
4.In a new test tube，add
0.1ml of the lymphocyte suspension
0.1ml of the absorbed and inactivated fetal calf serum
0.1ml 1%of the sheep erythrocyte suspension.
Mix well.Incubate at 37℃for 5 min.Then centrifuge at 500 rpm/min for 5 min.Put the test tube at 4℃for at least 2 h.
5.Take out the test tube.Discard part of supernatant.Mix well.Gently add a drop of 0.8%glutaraldehyde along the wall of test tube to fix cells.Put the tube at 4℃for 20 min.
6.Gently place a drop on a slide.Add a drop of 0.2% cold methylene blue.After 5 min，put a cover glass on it，observe and count the rossete forming cells under the microscope.
Results
If three or more erythrocytes are adherent to one lymphocyte，E-rossete formation is positive.The percentage of E-rossete formation is calculated by counting the number of rossete forming cells out of 200 lymphocytes.
Normal value：The percentage of E-rossete formation in human peripheral blood is 60%～80%.
Attentions
1.The sheep blood used in the E-rossete forming test must be fresh.It can be stored for less than two weeks.
2.Before the fetal calf serum is used，the complement must be inactivated by incubation at 56℃.
3.After the E-rossete formation，manipulation of the cells must be gentle to avoid separating the formed rossetes.