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Test 6 Specific cellular immune function assay

Test 6 Specific cellular immune function assay. Separation of Mononuclear Cells from Human Peripheral Blood Erythrocyte Rosette Forming Cell Assay Lymphocyte Proliferation Assay. Principle. Physical method density gradient centrifugation Most common method

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Test 6 Specific cellular immune function assay

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  1. Test 6Specific cellular immune function assay

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

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

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

  5. Methods 3ml veinous blood+ 3ml Hank’s solution 3ml diluted blood+2ml lymphocyte separation medium 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 suspension A drop 0.5% trypan blue Mix and stand for 5-10 min Draw a drop observe alive dead clear blue

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

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

  10. Lymphocyte Proliferation AssayPrinciple • A common method to measure cellular immune function in vitro • Nonspecific stimulators: PHA, ConA LPS

  11. PHA T B

  12. PHA刺激 48~72小时 • 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 calledlymphocyte transformation test.

  13. Morphologic counting Lymphoblast Mature lymphocyte transformed cells

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

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