**5. Structural tracking as a function of nanoparticle treatment**

Morphology modulations can be studied through documenting the changes in the cell as a dose and time dependent function of the exposing agent, using an inverted microscope [20]. Merged with Hoechst stained pictures, can provide additional information on the nuclear morphology. This blue fluorescent dye is membrane permeable and binds to DNA. Hoechst staining has also been successfully used to track changes in the nuclear integrity, thus documenting the progress of cell fate. Necrosis, apoptosis and cell enlargement have been successfully shown as consequences of different nanoparticle exposure in various cell-based studies.

as insoluble precipitates inside the cell. Cells are lysed, and these crystals are solubilized through combinations of various reagents such as detergent, DMSO, SDS, acidified isopropanol, dimethylformamide, etc. Absorbance readings are then documented, where maximum absorbance is proportional to higher survival. There are some negatively charges tetrazolium dyes such as MTS, XTT, and WST-1. These do not readily penetrate the cell. However, they

Resazurin (7-hydroxy-10-oxidophenoxazin-10-ium3-one) is particularly used to assay for mitochondrial dysfunction. Resazurin, a deep blue colored complex is reduced to resorufin, a pink colored complex by the enzymes in the inner mitochondrial membrane. Use of resazurin

ATP cell assay is yet another type of evaluation for cell viability. It overrides any incubation with live cell population. It is quicker, more sensitive and less prone to artifacts. Cells with damaged membranes cannot synthesize ATP and endogenous ATPases rapidly depletes cellular ATP concentration. Cells are lysed through detergent activity for this assay, in the presence of ATPase inhibitors to stabilize the total ATP content. Firefly or shrimp derived luciferase acts on substrate luciferin to generate light in the presence of ATP. Higher intensity

Sulforhodamine B (SRB) is a fluorescent dye [14]. It is bright pink in color. This aminoxanthene dye binds to cellular protein under mildly acidic conditions. Extraction in a basic environment is proportional to cell mass and thus an indicator of cell viability. Clonogenic cell survival assay investigates the cell's capability for propagation [15]. Treated sample of cells are plated and colonies stained and counted. DNA synthesis cell proliferation assay involves incubating cells with 3H-thymidine. Proliferating cells incorporates this radioactive tracker. Proliferation and thus cell viability can be measured by a scintillation counter. A non-radioactive alternative such as 5-bromo-2′-deoxyuridine (BrdU) can also be implemented. Although this includes an additional step of binding with BrdU-specific antibody and probably a secondary antibody before the colorimetric estimation [16]. A developed 5-ethynyl-2′-deoxyuridine (EdU) can be detected by a fluorescent azide through a Cu(I)-catalyzed cycloaddition reaction. This fast and sensitive method has additional advantages over BrdU assay [17], which are lack of sample fixation and preservation of DNA structure [18]. Raman micro spectroscopy detects variations in Raman bands associated with O–H stretching in water [19]. These variations can non-destructively correspond with ionic concentrations in the intracellular and extracellular fluids. This can be useful in determining the rate and direction of ionic transport. Loss of membrane integrity is dead and dying cells are often associated with leaching of ions, which

can still be used for the assay by incorporating an intermediate electron acceptor.

is inexpensive and more sensitive than using tetrazolium dyes.

of light is proportional to the number of live cells.

102 Cell Culture

can be detected and quantified by this technique.

**5. Structural tracking as a function of nanoparticle treatment**

Morphology modulations can be studied through documenting the changes in the cell as a dose and time dependent function of the exposing agent, using an inverted microscope [20]. Merged with Hoechst stained pictures, can provide additional information on the nuclear Phalloidin, a mushroom toxin, with a high affinity for F-actin can be used to track changes in actin dynamics [21]. Fluorescent probes are bound to phalloidin and thus help visualize the actin networks. Gap junctions in neuronal cells can be imaged by using biotinylated dextrans. Biotinylated dextran amines can be introduced in neural cell cultures by pressure injections [22]. Further they can be visualized by avidin conjugated horse radish peroxidase with a metal enhanced diaminobenzidine reaction.

Biocytin hydrazide, an aldehyde-based fixative, is another transneuronal tracer. It detects glycoconjugates and can be used to trace neuronal projections and visualize gap junctions. Biocytin hydrazide [23] in turn is detected by a fluorescent dye conjugated to streptavidin. Cholera toxin subunit B is a protein commonly used to retrograde tracer [24]. It binds to glycosphingolipids in axonal membranes [25]. It can also be used to visualize retinofugal projections. Subunit B is non-toxic and aids internalization and transport of conjugates. Wheat germ agglutinin (WGA) is a lectin protein that binds to N-acetyl-D-glucosamine and sialic acid [26]. Neurons can endocytose WGA-HRP, thus they can be used as tracers as they cross through synapses. Isolectins from legumes *Griffonia simplicifolia* can be used to differentiate between neuronal subtypes [27]. The A subunit prefers N-acetyl-D-galactosamine end groups while the B subunit is selective for terminal a-D-galactosyl residues. Its therefore used as a vascular stain for study of adult neurogenesis. Carbocyanine is a lipophilic dye used to stain plasma membranes [28]. They are highly fluorescent in lipid bilayers while being weakly fluorescent in aqueous phase providing a strong contrast for visualization.
