**6. Evaluation of key signaling pathways in studying cell fate**

Simple but conclusive evaluation of changes in expression of molecules is to conduct a reverse transcriptase polymerase chain reaction (RT-PCR) and carry out a western blot analysis. RT-PCR documents the changes in mRNA (transcriptional) levels, while western blot records changes at the protein (translational) level. Cells exposed to nanoparticles are harvested and thoroughly washed before downstream processing. RNA extracted is converted to cDNA and used as a template for PCR. Appropriate primers are designed for target amplification. Amplicons from the PCR are resolved using agarose gel electrophoresis and visualize by a UV illuminator using ethidium bromide staining.

For western blot analysis, protein is carefully extracted. Whole cell lysate (at least 40 μg) is resolved by SDS-PAGE [29]. Western blot is made by electrophoretic transfer on to membranes. Following transfer membranes are blocked with either non-fat dried milk or BSA. Overnight incubation with primary antibody is usually allowed. Post this, appropriate incubation is carried out with secondary antibody. Number of washes after each step, antibody dilution and incubation time all need to be optimized. Horse radish peroxidase conjugates are commonly used for color detection or chemiluminescent detection. However, chemiluminescent detection is much more sensitive than color detection. Expression of a target is normalized to an internal control such as β- Actin, to override experimental errors.

*In vivo* investigations are largely failing to correlate with clinical trials for various reasons. Many pharmaceutical companies have been using rat, monkey and dog models to evaluate toxicity. These are time consuming and cost intensive with very little productivity in terms of actual drugs reaching the market. *In vitro* investigations have the advantage of being cost and time effective and reduces unethical animal sacrifices and cruelty. Any meaningful result can

*In Vitro* Toxicity Testing of Nanomaterials http://dx.doi.org/10.5772/intechopen.80818 105

Another advantage of *in vitro* models is that it can be developed into 3D cultures and organ on chip innovations to more accurately predict clinical outcomes [32]. Some recent publications show that the level of prediction is even higher than that of *in vivo* investigations, since there is the scope to study and test human tissue functions along with the mechanical fluidic motions

Thus, it is highly intuitive to encourage young researchers to look at a problem holistically and design best possible routes towards solutions that may lead to implementation and relief. An appreciation of resources and time is vital to avoid wasting them in illogical endeavors. An open mind is to be inculcated that although is enriched with knowledge but chooses to

CMBL, Department of Biological Sciences, Birla Institute of Technology and Sciences, K K

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[2] Martin A, Sarkar A. Overview on biological implications of metal oxide nanoparticle exposure to human alveolar A549 cell line. Nanotoxicology. 2017;**11**(6):713-724

[3] Parasuraman S. Toxicological screening. Journal of Pharmacology & Pharmacothera-

[4] Merten O-W. Advances in cell culture: Anchorage dependence. Philosophical Trans-

[5] Abercrombie M. Contact inhibition and malignancy. Nature. 1979;**281**(5729):259

further be screened out for animal testing, if necessary.

that accurately mimic conditions in the human body.

\*Address all correspondence to: ansiemartin@gmail.com

and Drug Development Technologies. 2004;**2**(1):51-62

actions of the Royal Society B. 2015;**370**(1661):20140040

not be limited by it.

**Author details**

**References**

Ansie Martin\* and Angshuman Sarkar

peutics. 2011;**2**(2):74

Birla Goa Campus, Sancoale, South Goa, India

Different signaling cascade checkpoints and markers are commonly evaluated to understand the effect of the exposing agent on the cell through RT PCR and western blot analysis [30]. Some of these are highlighted. Akt, protein kinase B is involved in cell proliferation, transcription, migration and glucose metabolism. Caspases particularly, caspase 3 and caspase 9 indicate the progression of apoptosis. LC3B indicates the onset of autophagic processes. Hsp70 and Hsp90 indicate the heightened cellular responses of protein folding to external stresses. Expression of NFκB is an inflammatory response towards survival and cellular propagation. mTOR is directly or indirectly involved in the regulation of protein function.
