Dictyostelium Discoideum: Live Cell Imaging in Changing Perspective

The advent of advanced microscopes; during microscope evolution from simple microscopes to confocal and live cell microscope; having digital imaging facility revolutionized our view for the living cells. In the protein localization study, fluorescent proteins are tagged at amino or carboxyl (preferably) terminal of desired protein for live cell study. These live cell studies improved our understanding of protein dynamics and understanding its role in biological regulation. The mutational variants of fluorescent tags (GFP, RFP); can be used with different protein; which will efficiently use UV-Visible to Far Red light spectrum; without overlapping of excitation and emission spectrum. Further, various cell organelle (Lysosome, Golgi bodies, Endoplasmic Reticulum, Mitochondria, Nucleus) trackers; improved our live cell localization studies in the wide non-overlapping UV-Visible spectrum.This chapter gives an overview for live cell protein localization study in mitotically active, unicellular stage of Dictyostelium discoideum. This evolutionary cutting edge organism had both unicellular as well as multicellular stages during its life cycle. This chapter will provide the design of fusion of fluorescent tag to the specific gene and its live cell localization. Further, it will cover; transformation of the unicellular organism; drug based selection; sample preparation with nuclear, mitochondrial localization markers (trackers) and live cell localization study on live cell-confocal microscope setup. It will also have a glimpse of the design of fusion protein with an aspect of advantage and disadvantages.

Culture and Maintenance of Human Ovarian Carcinoma Cells for Scrutinizing Anti-cancerous Activities of Various Compounds via Some Potent Molecular Markers

Ovarian carcinoma is the 5th most common type of cancer of gynecologic origin and accounts for about one-fourth of the total malignancies of the female genital tract. Ovarian carcinoma accounts for highest mortality in females due to the development of chemo-resistance against drugs and lack of symptoms and undetectable biomarkers in the early stages of diagnosis. Tumour debulking, chemotherapies, radiotherapies, targeted therapies, immunotherapies and stem cell transplants are some of the measures that have been adopted by the experts for curing the disease but still, full control over the problem has not been achieved. Research on various herbal and chemosynthetic nano-compounds have shown a new light in this regard, as the studies on them so far have revealed that they have anti-proliferative and apoptotic properties that will help in finding new ways to develop drugs for cancer patients. This chapter deals how to culture and maintain the human ovarian carcinoma cell lines in the laboratory which are being procured from cell repositories and then to study the anticancer efficacy of various promising compounds by potent molecular markers like cellcycle progression and annexin V- FITC apoptosis detection.

2D-DIGE A Powerful Tool for Proteome Analysis

In the recent past, two dimensional gel electrophoresis has emerged as a powerful molecular biology tool for the comparative expression profiling of complex protein sample. It involves the separation as well as the resolution of diverse proteins sample on the basis of isoelectric points and molecular mass of protein in two dimension ways. In this way, it reflects the view of overall proteome status including differentiation in protein expression levels, post-translational modifications etc. Moreover, this allows the identification of novel biological signatures, which may give a particular identity of pathological background to cells or tissues associated with various types of cancers and neurological disorders. Therefore, by utilizing such tools, one can clearly investigate and compare the effects of particular drugs on cells of tissues and also one can analyze the effects of disease on the basis of variations in protein expression profile at broad spectrum. Recently, to get more error-less and accurate proteome profile, conventional 2-D gel electrophoresis has been enhanced with the inclusion of different types of protein labeling dyes which enables a more comparative analysis of diverse protein sample in a single 2-D gel. In this advanced technique (2-D-DIGE), protein samples are labeled with three different types of CyDyes (Cy2, Cy3, and Cy5) separately and combined and further resolved on the same gel. This will facilitate the more accurate spot matching on a single gel platform and will also minimize the experimental variations as commonly reported in the conventional 2D-gel electrophoresis. Therefore, in the present proteomic research era, 2D-DIGE has proved to be an extremely powerful tool with great sensitivity for up to 125 ng of proteins in clinical research volubility especially, neurological and cancer related disorders.

A Modified Western Blot Protocol for Enhanced Sensitivity in the Detection of a Tissue Protein

Western blots (WB) are designed to investigate protein levels and their patterns of modification in homogenized tissue samples. Although, Western blots are quantifiable, unlike immunohistochemistry, cellular integrity is lost. The availability of antibodies against the protein and their patterns of modification of interest form the basis of both Western blots and Immunohistochemistry. Antibodies can also be directed not only against proteins but against chemical modifications of the proteins too, such as phosphorylation and glycosylation of specific amino acid residues. In Western blotting, the proteins in the sample are denatured, size-separated on a denaturing acrylamide gel, and transferred to a nylon membrane. Antibody paratopes can then bind to the antigenic epitope in the protein present on the nylon membrane. Thus, with the help of a chemiluminescent assay system that darkens X-ray films, the resulting antibody-antigen complex can be visualized. Because of the ubiquitous and relatively inexpensive availability of WB equipment, the quality of WB in publications and following analysis and investigation of the data can be variable, possibly resulting in forged conclusions. This may be because of the poor laboratory technique and/or lack of understanding of the significant steps involved in WB and what quality control procedures should be followed to ensure effective data generation. The present book chapter focuses on providing a detailed description and critique of WB procedures and technicalities, from sample collection through preparation, blotting, and detection, to examination of the data collected. We aim to provide the reader with the improved expertise to decisively carry out, assess, and troubleshoot the WB process, in order to produce reproducible and reliable blots.

Analyzing Gene Expression through Real Time PCR while Neo-tissue Regeneration using Developed Tissue Constructs

Real-time PCR offers a wide area of application to analyze the role of gene activity in various biological aspects at the molecular level with higher specificity, sensitivity and the potential to troubleshoot with post-PCR processing and difficulties. With the recent advancement in the development of functional tissue graft for the regeneration of damaged/diseased tissue, it is effective to analyze the cell behaviour and differentiation over tissue construct toward specific lineage through analyzing the expression of an array of specific genes. With the ability to collect data in the exponential phase, the application of Real-Time PCR has been expanded into various fields such as tissue engineering ranging from absolute quantification of gene expression to determine neo-tissue regeneration and its maturation. In addition to its usage as a research tool, numerous advancements in molecular diagnostics have been achieved, including microbial quantification, determination of gene dose and cancer research. Also, in order to consistently quantify mRNA levels, Northern blotting and in situ hybridization (ISH) methods are less preferred due to low sensitivity, poor precision in detecting gene expression at a low level. An amplification step is thus frequently required to quantify mRNA amounts from engineered tissues of limited size. When analyzing tissue-engineered constructs or studying biomaterials–cells interactions, it is pertinent to quantify the performance of such constructs in terms of extracellular matrix formation while in vitro and in vivo examination, provide clues regarding the performance of various tissue constructs at the molecular level. In this chapter, our focus is on Basics of qPCR, an overview of technical aspects of Real-time PCR; recent Protocol used in the lab, primer designing, detection methods and troubleshooting of the experimental problems.

Osteosarcoma Cell Culture and Maintenance to Detect the Apoptotic Effect of Some Promising Compounds by Potent Markers viz. DNA

Osteosarcoma is the most common type of malignancy of bone and generally occurs among adolescent and young adults. Like the osteoblast cells of normal bone, osteosarcoma also forms the bone matrix, but the osteoid is not as strong as that of normal bones. Osteosarcoma is characterized by the production of weak or immature bones by the malignant cells. As the diagnosis of osteosarcoma is extremely poor, it suggests a critical need to develop some promising anti-osteosarcoma drugs to improve disease outcome. Many anti-cancer compounds induce apoptotic cell suicide via some potent cellular, molecular and biochemical markers. The cancer cell lines provide a valuable model system to study an extensive variety of cancer characteristics in the cell biology, genetics and chemotherapy or the impact of therapeutic molecules. The methods presented in this chapter describe the experimental technique used to culture the osteosarcoma cells for the documentation of DNA fragmentation and Caspase-3 activation associated with apoptosis.

Immunohistochemistry as an Important Technique in Experimental and Clinical Practices

Immunohistochemistry (IHC) is a well-known technique in the field of biological and medical sciences. This technique is based on the principle of antigenantibody interaction and is used for identification of cellular or tissue constituents, i.e., an antigen by using a specific antibody. The binding of an antibody to an antigen is confirmed either by labelled primary antibody itself or by using secondary labelling method such as fluorescence labelled antibody. Such interactions give information about the cellular process occurring inside the cell. In last few years, huge amount of data have been generated using IHC. Furthermore, adequate knowledge of this technique is required for the optimum result and its reproducibility. The detailed information about the tissue section, antigen retrieval (AR), increased sensitivity of the detection systems and proper standardization are the key points for this technique. This protocol will address overview of the technique, tissue preparation, microtome, antigen retrieval, antibodies and antigen fixation, detection methods, background reduction and trouble shootings.

Isolation of Genomic DNA From Plant Tissues

Genomic DNA extraction is the starting point for various downstream molecular biology applications viz. PCR, restriction analysis, hybridisation etc. Numerous problems like DNA degradation, co-isolation of viscous polysaccharides, polyphenols and other secondary metabolites causing damage to DNA, inhibiting restriction enzymes, DNA polymerases etc, are routinely encountered during DNA isolation from plants. Quinone compounds resulting from oxidation of polyphenols lead brown the DNA preparations and can also damage proteins and DNA’s due to their oxidizing properties. This results in a poor yield of high molecular weight DNA. The protocol below explains the extraction of DNA via the CTAB method, involving three major steps viz lysis of cell wall and membranes, extraction of genomic DNA and precipitation of DNA.