Cell isolation technique
Laboratory applications
Cellular immunity
A. Isolation of Mononuclear Cells from Peripheral Blood
Mononuclear cells in the peripheral blood can be separated from other cells by density gradient centrifugation.
A Ficoll-Hypaque solution (density of 1.077 g/L) is placed in a test tube. and diluted heparinized blood is layered on top.
The tube is then centrifuged. Low density cells (lymphocytes and monocytes) now float on top of the solution. and all other blood components form a pellet at the bottom of the tube.
The mononuclear cells at the Ficoll-plasma interface can be removed with a pipette. After incubation of the cell material in culture flasks, the monocytes adhere to the plastic walls of the bottle. This makes it possible to capture lymphocytes selectively.
B. Separation of T and B Lymphocytes (Rosette Formation)
T lymphocytes express adhesion molecules. such as the CD2 molecule. CD2 interacts with LFA 3 (CD58) on the surface of sheep red blood cells (SRBC).
After treatment with an enzyme........... such as neuraminidase or 2 aminoethyli sothiouronium bromide (AFT), the adhesion molecule on the RBC surface more readily inter acts with T lymphocytes.
Several SRBC can bind to a single Tcell. thereby forming rosettes." R0 sette forming cells can be isolated by density gradient centrifugation on Ficoll.
A fraction of approximately 95% pure T lymphocytes can be isolated after hypotonic lysis of the red blood cells. Cells that do not form rosettes float on top of the Ficoll layer and can also be isolated. These cells primath consist of B cells and other non T cells.
C. Antibody-Mediated Separation of Cell Fractions
Culture flasks or plastic dishes can be coated with antibodies at an alkaline pH (panning).
When a cell mixture is incubated on top of the antibody coated surface, the cells that express the target antigen will adhere to the plastic walls of the bottle or dish.
Antigen negative cells can easily be removed by decanting.
Antigen positive cells can be removed by mechanical means or by enzymatic digestion. By coating antibodies onto small beads that contain iron (available in different sizes).
it is possible to capture either antigen positive or antigen negative cell fractions. This method is called immunomagnetic separation. A magnet that attracts the iron loaded cells is used to separate antigen bearing cells.
This is a very effective method for removing unwanted cells from a solution. As little as a single contaminating tumor cell can be separated from 1000 nor mal cells by immunomagnetic separation. In other words. a 3 4 log depletion of unwanted cells can be achieved.
D. Cell Separation by Flow Cytometry
Normal flow cytometry was described as before .
A fluorescence activated cell sorter (FACS) is a specialized flow meter in which the fine cell-containing droplets are electrically charged under computer guidance.
Droplets with fluorescent cells are positively charged, and those with nonfluorescent cells (e.g.. anti gen negative cells) are negatively charged.
The stream of cells is guided through electric deilection plates. where the antigen positive and antigen negative cells are separated. then guided into separate collection tubes. The purity of cells separated by FACS is up to 99 %.
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