Encyclopedia of Experiments: Cancer Research
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Transcript
- T-cells, a class of lymphocytes, play an active role in fighting infection. Along with multiple other blood cell types, immunocompetent T-cells are present in mouse spleen and lymph nodes. To selectively isolate T-cells, harvest murine spleen and lymph nodes and macerate them on a cell strainer. Flush the tissue with an appropriate media to obtain a single cell suspension.
Add ammonium-chloride potassium, or ACK buffer, to lyse the red blood cells. Centrifuge to separate the intact blood cells from the lysate. Discard the supernatant and resuspend the cells in a suitable buffer. Treat the suspension with a mixture of biotinylated antimouse antibodies to target blood cells like granulocytes, B-cells, NK-cells, and any remaining erythrocytes. T-cells are not targeted and hence remain untagged.
Next, add anti-biotin magnetic microbeads to the suspension. All biotin-labeled cells bind to the microbeads. When kept under the influence of a strong magnetic field, the magnetically labeled cells adhere to the tube wall. Unlabeled T-cells remain in suspension. Collect the unbound enriched T-cells into a fresh collection tube. In the following protocol, we show T-cells' isolation from mouse spleen and lymph nodes, utilizing a magnetic separation technique.
- Pool the lymph nodes and the spleen in a 40 micrometer pore strainer and use a syringe plunger to macerate the tissues through the filters to release the cells. Wash the strainer and plunger with RPMI medium supplemented with 1% FBS to collect all of the cells and sediment the cells by centrifugation. Add 5 milliliters of ACK lysis buffer to the cell pellet.
After five minutes at room temperature, stop the reaction with 5 milliliters of medium supplemented with 1% FBS and pellet the white blood cells by centrifugation. Resuspend the pellet in 5 milliliters of MACS buffer for counting and dilute the cells to a concentration of 2 times 10 to the 8 cells per microliter in fresh MACS buffer. Add the appropriate concentration of anti-erythroid, anti-granulocyte, anti-B-cell, and anti-NK-cell depletion antibodies per 1 times 10 to the 6 cells for a 15-minute incubation at 4 degrees Celsius.
At the end of the incubation, wash the cells with 10 millimeters of ice-cold MACS buffer, and resuspend the pellet at a 1 times 10 to the 8 cells per milliliter concentration in fresh MACS buffer. Next, add 0.22 microliters of anti-biotin microbeads per 1 times 10 to the 6 cells with mixing for a 15-minute incubation at 4 degrees Celsius.
At the end of the incubation, wash the cells with 10 milliliters of ice-cold MACS buffer and collect the unbound enriched T-cells by magnetic bead separation using MACS express separator according to standard bead isolation protocols. At the end of the separation, sediment the T-cells by centrifugation and resuspend the pellet in 5 milliliters of fresh MACS buffer for counting.
