1. Test 6 Specific cellular immune function assay

2. Separation of Mononuclear Cells from Human Peripheral BloodSeparation of Mononuclear Cells from Human Peripheral Blood Erythrocyte Rosette Forming Cell Assay Lymphocyte Proliferation Assay

3. Principle Principle Physical method density gradient centrifugation Most common method Ficoll-Hypaque density gradient centrifugation

4. Ficoll-Hypaque mixed: 1.077 Lymphocytes and monocytes: 1.070 Granulocytes and erythrocytes: 1.092 centrifugation plasma Separation medium Granulocyte&Erythrocyte Lymphocyte & Monocyte

5. Methods Methods 3ml diluted blood+2ml lymphocyte separation medium 3ml veinous blood+ 3ml Hank’s solution Centrifuge 2000rpm, 20min Draw mononuclear cells +2ml Hank’s solution Centrifuge 1500rpm, 10min Discard supernant +1ml RPMI1640 culture medium Count and adjust cell concentration

6. 0.1ml cell suspensionA drop 0.5% trypan blue Mix and stand for 5-10 min Draw a drop observe alivedead clearblue

7. Attention Pay attention to aseptic manipulation when drawing peripheral veinous blood. Take care to protect yourself from blood borne infectious diseases. Complete the whole process in the shortest time to decrease the number of dead cells. It is necessary to use a horizontal centrifuge when separating PBMC with lymphocyte sepatation medium.

8. Erythrocyte Rosette Forming Cell Assay Principle Erythrocyte Rosette Forming Cell Assay Principle Erythrocyte Rosette Forming Cell Assay Erythrocyte Rosette Forming Cell Assay The test can be used to detect the number and ratio of T lymphocyte in peripheral blood. T T B SRBC CD2

10. Result The percentage of E-rosette forming lymphocytes that formed rosettes = Lymphocytes(formed + unformed rosettes) Normal value:60-80% X 100%

11. Lymphocyte Proliferation Assay Principle Lymphocyte Proliferation Assay Principle Lymphocyte Proliferation Assay Lymphocyte Proliferation Assay A common method to measure cellular immune function in vitro Nonspecific stimulators: PHA, ConA LPS

12. PHA PHA T B

13. When T lymphocytes are incubated with PHA in vitro, they are stimulated to increase the synthesis of nucleic acid and protein. The morphology of cells is transformed to a lymphoblast. This test is also called lymphocyte transformation test. PHA 刺激 48~72 小时

14. Morphologic counting Lymphoblast Mature lymphocyte transformed cells

15. Result The percentage of transformation ( transformed lymphocytes) X 100% = (Transformation+untransformed) lymphocytes Normal value:60-80%