3.2.3 Three-Dimensional Basement Membrane Assay
The 3D ECM plays an important role as a critical framework and cytokine immobilization scaffold for EC migration, connection, lumen formation and tube stabilization(Senger and Davis, 2011).By thickening the basement membrane or by adding an additional layer, the previously described 2D basement membrane assay can be easily converted into a 3D scaffold model(Deroanne et al., 1996).The 3D scaffold’s bottom, middle, or top can be covered with single cells or EC monolayers. On a fibronectin, laminin, or gelatin-coated dish, or at the surface of a collagen gel, ECs attach, spread, and multiply to form a monolayer. If the collagen layer is removed after covering with a collagen layer as a 3D matrix, ECs gradually organise into a network of tube-like structures, which then regress(Deroanne et al., 1996).Collagen type I and fibrin matrix appear to be particularly conductive to EC tube morphogenesis and sprouting(Davis and Senger, 2005). Fibroblasts cultured in the same conditions do not form a network, indicating that the 3D basement membrane is important for network formation as the specific behavior of ECs(Deroanne et al., 1996).When ECs are mixed into the 3D matrix, most of them participate in the morphogenic response. In contrast, in the case of the EC monolayer, only a subset of ECs participates in sprouting formation(Senger and Davis, 2011). The formation of an EC lumen in the 3D matrix has been observed and found to be dependent on the formation and coalescence of pinocytic intracellular vacuoles(Bayless and Davis, 2003). Compared to the 2D test, the 3D basement membrane inspection offers several advantages and is now one of the most used models for quantitative 3D angiogenesis in vitro(Bahramsoltani et al., 2009). It allows ECs to build structures that are not only capillaries, but lumens also. The movement of ECs in both horizontal and vertical aspects can be detected and analysed readily(Bayless and Davis, 2003).Fluidic flow is also applicable to understand the effect of shear stress on EC behaviour (Kang et al., 2008). However, the 3D basement membrane assay has limitations. These assays, for example, take significantly longer to run (5-15 days)(Staton et al., 2009).Furthermore, the 3D matrix thickness must be relatively small to allow oxygen and nutrients to diffuse while avoiding excessive cell mortality
4. Apoptosis assay (Annexin V-FITC)
This assay is performed by seeding the appropriate cell type and treatment with the test compounds (in serum free media) in their IC50 concentration for the predetermined time-points at 5% CO2 and 37 C in a humidified atmosphere. Untreated cells will be used as a negative control. Post-treatment with the test compounds, cells are harvested by trypsinization followed by PBS washing to clear off the cell debris. This step is followed by the addition of Annexin V-FITC, mixing and incubation in dark. The cells are then resuspended in binding buffer and incubated with propidium iodide (PI). The cells are analysed by flow cytometry(Kntayya et al., 2018).
5. Brine Shrimp Lethality Bioassay
The brine shrimp lethality assays are used to measure the cytotoxicity of the test compounds(Naher et al., 2019). In this experiment, brine shrimp eggs (Artemia salina) were incubated for 48 hours in a tank filled with sterile artificial seawater at 28–300 C with adequate aeration (using an air pump) and continuous light (60 W lamp). Following the collection of nauplii using a Pasteur pipette, brine shrimp are inserted into each well containing seawater. The serial dilution technique is used to create test solutions at various concentrations in individual vials filled with 5 mL of seawater containing 10 nauplii shrimps. After each vial has been examined under a microscope for 24 hours, the number of nauplii that survived is counted. The information is used to calculate the nauplii%, mortality of brine shrimp for both the control and increasing concentrations, and LC50 is calculated. The reference standard is potassium dichromate.(Lung and Cells, 2020).
Cell viability assays & cytotoxicity study
The different types of cytotoxic and cell viability assay include (a) dye exclusion assay such as Trypan blue, erythrosine B assay, (b) colorimetric assay such as MTT assay, MTS assay, LDH assay, SRB assay, NRU assay & crystal violet assay, (c)fluorometric assay such as Alamar Blue assay and CFDA-AM assay (d) luminometric assay such as ATP assay & real-time feasibility assay.