Encyclopedia of Experiments: Cancer Research
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Transcript
- Dendritic cells or DCs are key antigen presenting cells that activate T-cells. In leukemic patients, these cells poorly express tumor associated antigen and fail to elicit an antigen specific immune response. Dendritic cells are abundant in the bone marrow. And hence, the bone marrow is the preferred site for their isolation to help study their role in cancer.
To obtain dendritic cells, extract the bone marrow from a euthanized mouse's femur or tibia bone by using a media filled syringe to flush it out. Centrifuge and resuspended the pellet in ammonium-chloride-potassium or ACK buffer to lyse erythrocytes. Now add culture media to stop lysis and centrifuge. Resuspend the pellet in the culture media containing an antibiotic. Add granulocyte-macrophage colony-stimulating factor or GM-CSF, and transfer the cell suspension to a Petri plate.
Incubate the plate for the desired time duration. GM-CSF helps progenitor cells to differentiate into dendritic cells. Finally, activate the cells with lipopolysaccharides or LPS, which binds to the surface receptor of dendritic cells, resulting in fully mature and immunogenic bone marrow dendritic cells. In this protocol, we will demonstrate the generation of dendritic cells from the mouse bone marrow.
- To generate bone marrow-derived dendritic cells, isolate the bone marrow from the femurs of wild type or Factor B knockout mice, as demonstrated, and lyse the red blood cells in 5 milliliters of ACK lysis buffer, stopping the reaction with 10 milliliters of RPMI, supplemented with 1% FBS after five minutes.
After collecting the whole blood cells by centrifugation, resuspend the pellet in 10 milliliters of culture medium to a 2 times 10 to the 6 cells per milliliter concentration and add 20 nanograms per milliliter of GM-CSF to the cells, before seeding 10 milliliters of cells onto individual 100 by 15 millimeter Petri dishes at 37 degrees Celsius and 5% carbon dioxide for six days. On day six, activate the bone marrow-derived dendritic cell cultures with 25 micrograms per milliliter of LPS per plate.
