Human Peripheral Blood-Derived Monocytes

General Description Specifications Certificate Of Analysis Technical Library

Human peripheral blood derived monocytes are a type of white blood cells that originate in bone marrow and circulate in the bloodstream. These monocytes play a very important role in the development of the innate immune system and are responsible for phagocytosis, or the engulfing and destruction of pathogens. They are also responsible for presenting antigens to T cells to activate the adaptive immune response. Research is ongoing to understand their role in viral pathogenesis and development of inflammatory diseases like rheumatoid arthritis, atherosclerosis, etc. Studies are ongoing to investigate the molecular mechanism by which monocytes are recruited to the site of inflammation and the factors that are responsible for regulation of macrophages and dendritic cells. Their role in cancer immunotherapies has also been recently explored, in order to evaluate their potential role in the development of cancer vaccines. Kosheeka Human Peripheral Blood Derived Monocytes are isolated from peripheral blood of screened, healthy, untreated donors. Briefly, mononuclear cells are isolated from apheresis derived peripheral blood through differential centrifugation methods. The population of mononuclear cells is further purified through positive immunomagnetic selection directed against CD14. These cells are further characterized for the expression of CD14 markers. The donor blood is also screened for infectious markers like HIV, HCV, HBsAg and HBcAg.

Product Category	Homo Sapiens, Human
Product Type	Adherent, anchorage-dependent population
Derived From	Peripheral Blood
Cell Morphology	Round shaped
Culture & Growth Properties	Doubling time of approximately 20hrs.
Passage No.	P1/P2
Mycoplasma	Detected on demand
Hepatitis B	Negative
Hepatitis C	Negative
HIV-1	Negative
HBsAg	Negative
HBcAg	Negative
Aerobic & Anaerobic Bacteria	Negative after 7 days of incubation
Positive for	CD14
Negative for	CD34

The monocytes were first described by Metchnikoff in the year 1882 and are demonstrated to be critically important in regulating immune cells during normal and diseased condition. These cells are identified to be leukocyte subset that play a key role in maintaining tissue homeostasis, pathogen challenge and clearance. In humans, these cells represent approximately 10% of the total concentration.

The Kosheeka CD14+ cells are isolated from a mononuclear fraction of peripheral blood from healthy donors through a magnetic cell separation method. The method comprises positive selection of CD 4 cells tagged with specific magnetic beads. The blood cells are passed through the appropriate magnetic field, which selectively pulls the magnetically labelled monocytes towards the magnet, allowing them to be collected. However, if our client wants us to separate them with some other method like density gradient centrifugation or fluorescence activated cell sorting etc. we can customize our separation method as well.

Monocytes can be cultured in a variety of media depending on the experimental requirements. However, some of the common media that are generally used for culturing peripheral blood derived monocytes are RPMI 1640, Dulbecco’s modified eagle’s medium (DMEM) and M199. These media are generally combined with suitable growth factors, cytokines and 10% fetal bovine serum to optimize cell growth. To improve adherence, we also recommend use of 10ng/ml of M-CSF; however, it should be noted that adhered monocytes are destined to become macrophages.

Yes, these cells can be differentiated into macrophages by adding suitable concentration of M-CSF. Kosheeka has validated the protocol by adding 10ng/ml of M-CSF in medium, while cells are allowed to grow on poly-D lysine coated plates. However, it is important to validate your own protocol.

The choice of growth factor depends mostly on your experimental conditions. However, variety of growth factors are generally used during culturing peripheral blood derived monocytes, such as macrophage colony stimulating factors (M-CSF), granulocyte macrophage colony stimulating growth factors (GM-CSF), interleukin 4 (IL-4), interleukin 6 (IL-6), Tumor Necrosis Factor (TNF-alpha) and certain lipopolysaccharides.

The doubling time of monocytes can vary depending upon the various factors like culture condition, donor availability and the presence of specific growth factors. In general, the doubling time of monocytes in culture is relatively slow as compared to other cell types and is found to be around 30-60 hrs. Several studies have reported the doubling time of monocytes in different culture conditions. For instance, in the presence of M-CSF, the doubling time of monocytes has been reported to be around 40-48 hrs, while in the presence of GM-CSF, the doubling time is reported to be 34-44 hrs. On the other hand, the choice of media also defines cell doubling time, like with serum free media, the doubling time can be little longer as compared to media with appropriate concentration of fetal bovine serum. Importantly, depending upon culture conditions and experimental requirements, the doubling time of monocytes should be validated.

The monocytes are transported in suitable culture media like RPMI or DMEM along with specific supplements like cytokines, growth factors etc. for optimum culture conditions. These cells are typically transported in temperature-controlled container to maintain their viability. On demand, we can also ship cells in phosphate buffer saline, supplemented with appropriate concentration of BSA in a temperature-controlled manner. We can recommend different choices of transportation depending upon specific experimental requirements, the distance of transportation and the resources available.

Yes, monocytes can further be passaged in culture, although their proliferative capacity is generally limited. After differentiation to dendritic cells and/or macrophages, which have higher proliferative capacity; these cells can be passaged further with the addition of suitable growth factors and/or cytokines. Some of the general steps that can be followed for passaging monocytes are detach the monocytes using gentle cell scraper, count the number of cells per ml and appropriately plate approximately 1000 cells per well coated with either fibronectin, collagen or poly-D lysine to promote cellular attachment and proliferation. In order to promote proliferation rate, certain growth factors like M-CSF or GM-CSF are recommended depending upon culture conditions. The cultured cells need to be observed daily for growth and confluence.