Encyclopedia of Experiments: Immunology
A subscription to JoVE is required to view this content.
Transcript
When all of the bones have been collected, place the samples in PBS on ice, and transfer the samples to a sterile culture hood. Using sterile technique, make 1 to 2-millimeter cuts at both the proximal and distal ends of each bone before placing three bones into each modified micropipette tip in the microcentrifuge tubes.
Harvest the bone marrow from the bones by centrifugation, confirming that the marrow has been completely pelleted to the bottom of each microcentrifuge tube at the end of the spin. If all of the marrow has been collected, the bones will appear white. Remove the micropipette tips and the bones from the collection tubes for proper biosafety disposal.
To plate the bone marrow cells, first use a 1-milliliter syringe equipped with a 25-gauge needle to slowly resuspend the pellets and to break up any clumps, and pool the samples into a single 15-milliliter conical tube. Add 10 milliliters of bone marrow stem cell culture medium per 500 microliters of sample, and filter the cell suspension through a 70-micron filter into a 50-milliliter conical tube to remove any bone fragments.
To plate a split bone marrow cell population culture, plate the harvested bone marrow cells from one mouse in a 10-centimeter cell culture dish for 48 hours in fresh culture medium in a cell culture incubator.
On day two of culture, remove the non-adherent hematopoietic stem cell-containing supernatant, and carefully wash the plate with PBS without disturbing the adherent bone marrow cells. Replace the PBS with 0.25% trypsin. After 1 to 3 minutes at 37 degrees Celsius, quench the reaction with fresh cell culture medium, and count the cells in the resulting cell suspension.
Centrifuge the appropriate number of cells for plating, and resuspend the pellet in enough fresh culture medium for the desired plating density. Then, place the plates in the cell culture incubator until confluency.
