Manual lymph drainage can increase the rate of lymph drainage in the body by 20 times. Lymphatic massage is a specialized massage to increase this lymph drainage. It is not a normal massage which mainly massages muscles. A vision is most of all about understanding your customers. To watch the GRIPR instruction video, visit. PAMBR05V2: Size 1479mm x 400mm x 620mm. Body workout system designed to be the most. The Octagon Bench is designed to provide avid strength-trainers. Boosted lymphatic drainage.
. 10k Downloads. Abstract The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays.
These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference. Introduction 2. Endothelial cell and monocyte migration assays 3.
Endothelial cell proliferation assays 4. 3D models of vascular morphogenesis 5.
Aortic ring assay 6. Tumor microvessel density and histopathological growth patterns in tumors 7. Assessment of intussusceptive angiogenesis 8.
In vivo sprouting lymphangiogenic assay and AAV-mediated gene transfer of vascular endothelial growth factor c (VEGFC) 9. Assay for pericyte recruitment to endothelial cell-lined tubes, capillary assembly, and maturation 10. EC co-culture spheroids 11.
Endothelial cell metabolism 12. Endothelial cell precursors 13.
Microfluidic assays 14. Flow cytometry and cell sorting assays 15. Loss-of-function approaches in the developing zebrafish 16.
Chorioallantoic membrane assays 17. Murine allantois assay 18. In vivo angiogenesis plug assay 19. In vivo vascular network forming assay 20. Developing mouse retinal vasculature—tip cells 21. Corneal angiogenesis assays 22. Mouse oxygen-induced retinopathy model 23.
Laser-induced choroidal neovascularization mouse model 24. Transparent window preparations for angiogenesis studies in mice 25. The RIP1-Tag2 transgenic mouse model 26.
The MMTV-PyMT breast cancer model 27. Tumor implantation models 28. Mouse hind limb ischemia model 29. Large animal models for myocardial angiogenesis 30.
Guidelines for purity of recombinant proteins in angiogenesis assays 31. Conclusions Introduction.
The process of angiogenesis—the formation of new blood vessels from preexisting ones—is a hallmark of tissue repair, expansion, and remodeling in physiological processes, such as wound healing, ovulation, and embryo development, and in various pathologies including cancer, atherosclerosis, and chronic inflammation ,. Many of these conditions share characteristics, for example the occurrence of hypoxia or inflammation, recruitment of inflammatory cells, angiogenic growth factor production, basement membrane degradation, endothelial cell (EC) migration, proliferation and differentiation, and modulation of vascular support cells. However, depending on the tissue or disease under investigation, important details may differ considerably. Moreover, EC in different vascular beds exhibits organ-specific heterogeneity associated with the differentiated specialized functions of the tissue. It is often not possible to accurately visualize the process of angiogenesis and its molecular players.
Therefore, different in vivo, ex vivo, and in vitro bioassays and techniques have been developed to investigate the specific stages of the angiogenesis. However, the use of bioassays that study a part of the process, with the intention to extrapolate and understand the full process of angiogenesis, inherently implies accepting specific limitations. It is therefore crucial to understand the full potential of these bioassays during their specific applications. These assays have been instrumental in the study of vascular biology in growth and development but also play a key role in the design, development, and evaluation of drugs that positively or negatively modulate vessel function for the treatment of many diseases ,. EC migration is one of the hallmarks of angiogenesis and one of the earlier steps in the angiogenic cascade.
This process is characterized by cell-autonomous motility property but in some cases, it acquires the features of collective migration , , in which a group of cells coordinate their movements toward a chemotactic gradient and by establishing a precise hierarchy with leader and follower cells. Therefore, dissection of the molecular mechanisms of EC migration is critical to understand and to therapeutically manipulate the process to either inhibit sprouting (e.g., in tumors) or stimulate vessel formation (e.g., during tissue regeneration or wound healing).
Likewise, migration assays have been successfully used to assess the migratory responsiveness of monocytes. Monocytes are actively involved in angiogenesis, and their migratory response or potential correlates well with that of endothelial cells. Most importantly, CD14-positive monocytes can easily be isolated and obtained from any individual, not only humans , , but also mice. A number of 2D and 3D cellular migration assays have been established as relatively simple in vitro readouts of the migratory/angiogenic activity of EC in response to exogenous stimuli. Depending on the specific scientific question, a range of assays is available to quantitatively and qualitatively assess EC migration. The most widely employed assays include variations of the wound closure and the Boyden chamber assays.
Types of assays Cell culture wound closure assay Lateral migration assays are performed to investigate the pro- or anti-migratory effect of compounds, as well of specific gene perturbations, or to describe phenotypes resulting from genetic manipulation of EC. Although these assays can be used to characterize chemokinesis (unidirectional migration) in response to a given compound added to the cell culture medium, they do not allow determination of directed migration rate toward or away from a compound. Assessment of chemotaxis can only be determined when a gradient is also provided. The cell culture wound closure assay is one of the basic readouts for characterizing the migratory activity of cells.
It is a measure of the lateral 2D migration of EC in cell culture to test compounds for pro-migratory or anti-migratory activity. Depending on the migratory effect of the tested substances, the assay is performed over 2–4 days. ECs are grown to confluency in a cell culture dish and then scraped with a razor blade/pipette tip , allowing the EC at the wound edge to migrate into the scraped area.
To really examine the motility contribution to the healing and to exclude the component related to cell proliferation, ECs are incubated with the antimitotic agent mitomycin. Large genome-wide screens can also be assessed with the scratch wounding technique. The use of precision wounding replicators with floating pins and a workstation robot enables large numbers of scratches to be made with reduced coefficients of variation ,. Wound healing assay connected with video-lapse microscopy allows studying in 2D dimension the role of collective migration in angiogenesis and vascular development ,. The use of aortic rings (see below) and that of specific microfluidic devices represent a further tool to describe this process in a 3D architecture. Many regulators of angiogenesis have been identified, validated, and developed based on their effects on EC proliferation.
ECs are among the most quiescent cells in the body, with proliferation rates approaching zero under steady-state conditions. Only after stimulation, usually as a consequence of injury, inflammation, or pathological processes such as malignant growth, can they initiate cell cycle entry ,. The ideal assay to measure EC proliferation should be rapid, reproducible, and reliable and wherever possible should exclude inter-operator variability, for example through quantitative computational readout rather than qualitative researcher-dependent observations. This section presents different methods and elaborates on problems and pitfalls. Types of proliferation assays. A number of different approaches to address cell proliferation have been developed in the last decades. In general, these include assessment of cell number, detection of DNA synthesis by incorporation of labeled nucleotide analogs, measurement of DNA content, detection of proliferation markers and metabolic assays (Fig.
Depending on the broadness of the definition of cell proliferation, which can range from the narrow description, for example, “the fraction of cells dividing over time” to the more general “the doubling time of a population,” several different assays may be pursued. Apart from that, means and equipment available will also dictate the choice of a particular method. As all methods focus on a particular aspect of the process, it is highly recommended to verify results with a complementary assay. Fig. 1 Endothelial cell proliferation assays.
A Phase-contrast image (left) and binarized image of HUVEC grown in a regular 96-well plate. Simple software solutions can be used to count features in the image.
B Example of MTT assay, with color intensity correlating with cell number. C DNA staining profile of HUVEC using PI, measured on a plate cytometer. D Cell viability of HUVEC exposed to sunitinib, measured using a luminescent assay Cell counting Cell counting is considered the gold standard for proliferation.
![Body vision 620 weight bench manual lymphatic drainage machine Body vision 620 weight bench manual lymphatic drainage machine](/uploads/1/2/5/4/125480329/331651334.png)
Moreover, at least in theory, it is one of the most straightforward procedures for measuring proliferation of a cell population. It can be done using automated cell counters (e.g., Beckman Coulter) or by using a hemocytometer after removal of the cells from the culture vessel ,.
More recently, different automated platforms have entered the market that allow analysis of cells while present in microplates, such as plate cytometers, automated microscope, or high-content screening platforms, that are compatible with cell counting-like procedures. With these, cells can be monitored over time but frequently require staining for detection, calibration, and (computation-assisted) quantification by, for example, staining of nuclei.
Moreover, real-time cell analysis (referred to as RTCA) platforms have emerged that allow label-free, automated, real-time monitoring of cellular properties during incubation based on electrical resistance measurements. The equipment, however, requires considerable investment, beyond reach for many laboratories. DNA labeling During S phase of the cell cycle, DNA is synthesized and subsequently divided between the daughter cells (2 N → 4 N → 2 N; N = number of a complete set of chromosomes).
Addition of modified nucleotides to the culture medium will result in their incorporation into the newly synthesized DNA. Adhering to the narrow definition of proliferation as stated above, this type of assay most closely reflects a means of measuring the fraction of actively dividing cells. It should be noted that this technique does not directly measure cell division or population doublings, but exclusively incorporation of a tracer into DNA synthesis. 3H-thymidine has been used in proliferation assays for decades ,. Briefly, cells are pulsed with 3H-thymidine for several hours and radioactivity is measured by liquid scintillation counting. This provides a very accurate representation of DNA synthesis and it is highly sensitive since the amount of incorporated 3H-thymidine is directly proportional to the rate of DNA synthesis ,. Constraints on using radioactive compounds and the rise of alternative methods have limited its use.
In a similar manner, the incorporation of 5-bromo-2′-deoxyuridine (BrdU) or EdU (5-ethynyl-2′deoxyuridine) can be measured. BrdU or EdU can be (in)directly detected and subsequently be (semi-)quantified using ELISA, flow cytometry, or immunohistochemistry ,. The latter two quantification techniques allow one to determine the fraction of dividing cells. These uridine analogs can be combined with DNA dyes (see below) to gain additional cell cycle information. Another approach is the measurement of cellular DNA content using intercalating dyes such as PI (propidium iodide) or DAPI (4′,6-diamidino-2-phenylindole).
Using flow or plate cytometry, a profile of the distribution of cells over the different phases of the cell cycle can be visualized, represented by DNA contents of 2 N (G1/0), 4 N (G2/M), or mixed (S). In addition, this method allows for the detection of apoptotic cells that would exhibit a subG1/0 (.