oren-knox
Uploaded by
15 SLIDES
679 VUES
200LIKES

Test 6 Specific cellular immune function assay

DESCRIPTION

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

1 / 15

Télécharger la présentation

Test 6 Specific cellular immune function assay

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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%

More Related