Culturing and Harvesting of HSPC Clones: A Method to Obtain the Singly-Sorted HSPC Clones

- Hematopoietic stem and progenitor cell or HSPC, can self-renew and differentiate into various types of blood cells. HSPCs gradually accumulate DNA mutations during a lifespan, which can contribute to leukemia. To analyze such mutations, HSPCs are generated usually from the bone marrow.

Begin by adding fluorophore-conjugated antibodies to mononuclear cells-- adult stem cells isolated from the bone marrow-- and incubating them for the desired duration. The antibodies bind to specific cell surface markers such as CD34. Now, load the mixture in the fluorescence-activated cell cytometry or FACS machine.

The fluorophore-conjugated antibodies fluoresce when hit by the laser. Individual cell droplets pass through the detector that measures the fluorescence intensity, based on which the drop possesses an electric charge. In the presence of electromagnetic fields, positively charged cells move to the negative container and vice versa.

Now, grow the FACS-sorted CD34-positive HSPCs in an appropriate medium for the desired duration until clones are visible. Next, dislodge the clones by vigorous pipetting and centrifuge to pellet the cells. Store the pellet containing clonal HSPCs at minus 20