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.
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- Detection method
- Fluorescent
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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.
Key facts
- Detection method
- Fluorescent
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- Multi
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Notes
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.
The Hematopoietic Colony Forming Cell Assay Kit (ab242288) 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 GR Dye in a fluorescence plate reader (see Assay Procedure 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.
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- Activity summary
Hematopoietic stem cells (HSCs) are a type of stem cell that give rise to all blood cell types including myeloid cells (such as monocytes macrophages and neutrophils) and lymphoid cells (such as B and T lymphocytes). These cells are defined by their capacity for self-renewal and multipotency. HSCs can be found in the bone marrow blood and umbilical cord blood. They are not characterized by a single marker but a combination such as CD34+ CD38- and CD90+ in human cells. The term HSC is an alternate name often synonymous with hematopoietic colony-forming units. The typical mass of a singular HSC is not defined as it varies due to cellular conditions and differentiation states. Various methodologies like colony-forming assays and hematopoietic colony counters measure the functional capability of these cells to form colonies in vitro.
- Biological function summary
Hematopoietic stem cells function as the cellular basis for the entire blood system. They reside in a specialized microenvironment called the niche which regulates their behavior and interactions with supporting stromal cells and factors. This intricately balanced system ensures homeostasis of blood cell production and responds to the body's needs. HSCs often associate with cellular populations in bone marrow contributing to blood cell lineage differentiation through interaction with cytokines and other signaling molecules. Biologists commonly use assays such as the CFU assay protocol and CFU assay kit to test for hematopoietic potential in both research and clinical settings.
- Pathways
Hematopoietic stem cells operate predominantly within the hematopoiesis and Notch signaling pathways. Within hematopoiesis they play a central role in the continuous production of blood cells and sustain lifelong hematopoiesis by changing activity states in response to different signals. The Notch signaling pathway regulates the cell fate decisions of HSCs influencing their self-renewal and differentiation and interacts with various proteins such as Delta and Jagged ligands. An understanding of these pathways is important for deciphering how HSCs maintain bone marrow homeostasis and support immune system functionality.
- Associated diseases and disorders
Hematopoietic stem cells have relevance in conditions like leukemia and anemia. Leukemia involves abnormal proliferation and differentiation of blood cells and is often associated with genetic or environmental changes affecting HSCs or their niches. Anemia can arise from insufficient production or loss of red blood cells sometimes linking to malfunctioning HSCs. The protein FLT3 often connects with HSCs in leukemia where its mutations can lead to uncontrolled cell growth. Understanding the role of HSCs in these diseases enables development of targeted therapies such as stem cell transplantation which seeks to replace malfunctioning cells to restore blood health.
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3 product images
Functional Studies - Hematopoietic Colony Forming Cell Assay Kit (ab242288)
CD34+ Cell Dose Curve.
Human bone marrow derived CD34+ Hematopoietic Progenitor Cells were resuspended at 2 x 106 cells/mL and titrated 1:2 in resuspension medium, followed by addition of Cell Lysis Buffer, and GR Dye detection (as described in the Cell Dose Section).
Functional Studies - Hematopoietic Colony Forming Cell Assay Kit (ab242288)
Colony Formation.
Human bone marrow derived CD34+ Hematopoietic Progenitor Cells were seeded at 3000 cells/well and cultured for 7 days in the presence or absence of growth factors/cytokines (50 ng/mL SCF, 10 ng/mL hIL-3, 10 ng/mL hGM-CSF, 3 U/mL hEPO). Colony quantitation was determined according to the assay protocol. Photographs were taken after 7-day culture for Panel A (without growth factor/cytokine supplement) and Panel B (with growth factor/cytokine supplement). Panel (C) demonstrates growth after 10 days with growth factors/cytokines (hemoglobin clearly visible).
Functional Studies - Hematopoietic Colony Forming Cell Assay Kit (ab242288)
Colony Formation.
Human bone marrow derived CD34+ Hematopoietic Progenitor Cells were seeded at 3000 cells/well and cultured for 7 days in the presence or absence of growth factors/cytokines (50 ng/mL SCF, 10 ng/mL hIL-3, 10 ng/mL hGM-CSF, 3 U/mL hEPO). Colony quantitation was determined according to the assay protocol.
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