病原微生物及免疫學(xué)融合實(shí)驗(yàn)：Specificcellular immune function assay

1、Separation of Mononuclear Cells from Human Peripheral BloodErythrocyte Rosette Forming Cell AssayLymphocyte Proliferation AssayImmunology Experiment 1 -Specific cellular immune function assay PrinciplePhysical method density gradient centrifugationMost common methodFicoll-Hypaque density gradient ce

2、ntrifugation Ficoll-Hypaque mixed: 1.077 Lymphocytes and monocytes: 1.070 Granulocytes and erythrocytes: 1.092centrifugationplasmaSeparation mediumGranulocyte&ErythrocyteLymphocyte & Monocyte Methods3ml diluted blood+2ml lymphocyte separation medium1.5 ml veinous blood+ 1.5 ml Hanks solutionCentrifu

3、ge 2000rpm, 20minDraw mononuclear cells +2ml Hanks solutionCentrifuge 1500rpm, 10minDiscard supernant ，put 1ml RPMI1640 culture mediumCount and adjust cell concentration0.1ml cell suspensionA drop 0.5% trypan blueMix and stand for 5-10 minDraw a dropobservealivedeadclearblueAttentionPay attention to

4、 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 sepa

5、tation medium. Erythrocyte Rosette Forming Cell AssayPrincipleThe test can be used to detect the number and ratio of T lymphocyte in peripheral blood. TTBSRBCCD2ResultThe percentage of E-rosette forming lymphocytes that formed rosettes= Lymphocytes(formed + unformed rosettes)Normal value: 60-80%X 10

6、0% Lymphocyte Proliferation AssayPrincipleA common method to measure cellular immune function in vitroNonspecific stimulators: PHA, ConA LPS PHATBWhen 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

7、transformed to a lymphoblast. This test is also called lymphocyte transformation test. PHA stimulating4872 hMorphologic counting Lymphoblast Mature lymphocyte transformed cellsResultThe percentage of transformation ( transformed lymphocytes) X 100%= (Transformation+untransformed) lymphocytesNormal value:60-80%3. Methods: RBCWBCWBCWBCWBC1mm1mmCell concen