Description
• Methylcellulose medium supports growth of hematopoietic cell colonies
• Fully quantify hematopoietic stem cells with no manual cell counting
• Results in 7-10 days, not 3 weeks
Hematopoietic stem cells (HSCs) are well-characterized, tissue-specific stem cells that are responsible for the lifelong maintenance of the hematopoietic system. HSCs or hematopoietic progenitors known as colony-forming cells (CFCs) proliferate to form discrete colonies when cultured in a suitable 3D environment, such as methylcellulose supplemented with nutrients and cytokines. Our CytoSelect 96-Well Hematopoietic Colony Forming Cell Assay promotes the formation of HSC colonies in just 7-10 days. Cells can then be either quantified in a fluorescence plate reader or recovered from the semisolid medium for further downstream analysis.
Introduction: Hematopoietic stem cells (HSC) are well-characterized, tissue-specific stem cells that exhibit remarkable self-renewal capacity and are responsible for the life-long maintenance of the hematopoietic system. HSC are rare cells that reside in adult bone marrow where hematopoiesis is continuously taking place, but can also be found in cord blood, fetal liver, adult spleen and peripheral blood. Throughout the life span, hematopoiesis continuously replenishes the various lymphoid, myeloid and erythroid-megakaryocyte lineages, but also to maintain a small pool of HSC with the self-renewal capacity that is capable of carrying on hematopoiesis. From HSC to mature blood cells, extensive proliferation and expansion occurs that results in the production of millions of blood cells.
When cultured in a suitable semi-solid matrix, such as methylcellulose supplemented with nutrients and cytokines, HSC or hematopoietic progenitors called colony-forming cells (CFCs) proliferate to form discrete cell clusters or colonies. Colony types include colony-forming unit-erythroid (CFU-E), burst-forming unit-erythroid (BFU-E), CFU-granulocyte, macrophage (CFU-GM) and CFU-granulocyte, erythrocyte, macrophage, megakaryocyte (CFU-GEMM).
In classical CFC assays, culture usually takes place in a 35 mm dish for 14-21 days for the colonies to reach certain size (> 40 cells/colony) for manual counting. The CFCs are classified and manually enumerated based on the morphological recognition of one or more types of hematopoietic lineage cells within the colony.
MyBioSource's CytoSelect 96-well Hematopoietic Colony Forming Cell Assay does not involve subjective manual counting of colonies or require a 2-3 week incubation period. Instead cells are incubated only 7-10 days in a semisolid methycellulose media before being solubilized, lysed and detected by the patented CyQuant GR Dye in a fluorescence plate reader (see Assay Principle below). Alternatively, viable CFCs can be easily recovered for further culturing and testing. This format provides a quantitative, high-throughput method to accurately measure HSC or hematopoietic progenitor clonogenic capability