Interdiction for experiments - Assignment Example

A conventional cell culture media generally consist of vitamins, glucose, well balanced amino acids, serum as a source of growth factors, hormones as energy source for the regulation of cell cycle (Waymouth, 1972). Osmolality and pH are the other two aspects which govern the environment of cell culture. pH 7.4 has been proved as the optimum pH to grow mammalian cells in culture. Although small deviations exist. The culture medium acts as a buffer and inhibits the pH change. As bicarbonate and dissolved CO2 has an impact on the pH of the medium, atmospheric CO2 also plays a crucial role in maintaining the pH of the medium.

Therefore CO2 is used form a exogenous source and in the incubator it is generally maintained at 5%. For most of the culture conditions the temperature is maintained at 37°C for normal growth of the culture cells. Finally, osmolality is maintained in culture condition since at 37°C the culture media can be evaporated. Hence to prevent the evaporation of the culture media, humidifying condition is maintained by keeping water in the incubator. A failure in controlling the culture environment leads to contamination of culture, unhealthy cells, more apoptotic cells and other abnormal morphologies (Waymouth, 1970).

What is the fluorescence-activated cell sorting, why it is important? Flow cytometry or fluorescence activated cell sorter (FACS) is generally used to examine the microscopic particles like cells. First the cells are suspended in a fluid and then a stream of fluid containing the cell suspension is passed over a laser beam. This results in light scattering and fluorescence emission. The next step is the filtration and collection of the scattered and emitted lights which are then converted to digital signals.

These signals are then analyzed by appropriate software. Thus the fundamental basics of flow cytometry comprises of fluidics (cell suspension and the hydrodynamic focusing of the fluid), optics (laser beam, light scattering and fluorescence emission) and electronics (conversion to digital values and output through computer). By FACS one can analyze multiparametric physical and chemical behavior of cells. It allows the analysis of thousands of particle in a second. The stream of fluid is hydrodynamically focused and the beam of light usually a laser is pointed towards the stream of fluid.

Two different types of detectors are placed to detect the scattered light. One is in line with the laser beam known as forward scatter (FSC) and many detectors are in perpendicular with the laser known as side scatter (SSC). FSC is attributed to the size of the particle where as the SSC refers to the granularity that is the internal complexity of the particle (Ormerod 2000). Fluorescence-activated cell sorter is generally used in biomedical science which includes the fields like pathology, transplantation, hematology, immunology, molecular biology, signal transduction.

Various parameters can be ascertained by FACS. Some of which are- cell cycle and tumor ploidy (El-Naggar 2004), immunophenotyping, ion flux, membrane potential, intracellular protein staining, cell proliferation, pH changes (Rabinovitch and June 2000), cell viability, cell sorting, chromatin structure, redox state, total protein, lipids, surface charge, enzyme activity, gene expression and DNA degradation (Darzynkiewicz et al 1997) What is the Immunoprecipitation Immunoprecipitati