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Cell Biology Lab.docx

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Lab 2 Cell culture of C2C12 mouse myoblast cells Examination of the cells under phase microscope : Color = grey ; Shape= circular rod like PBS( Phosphate buffered saline ) - Is a water based solution containing sodium chloride and sodium phosphate and it helps to maintain constant pH. EDTAin PBS is used to disengage attached and clumped cells. In order to reduce any possible contamination of the cell culture there are sterilization techniques that are employed in the lab such as performing the cell culture in a room designed to be sterile and working in the laminar fume hood .The flow of air protects the area from dust and contamination and acts as a barrier between the work surface and the worker.Also the Ultraviolet light helps to reduce the decontamination of the work surfaces. C2C12 cells in horse serum become differentiated . Trypsin In a tissue culture lab, trypsin are used to re-suspend cells adherent to the cell culture dish wall during the process of harvesting cells. Some cell types have a tendency to "stick" - or adhere - to the sides and bottom of a dish when cultivated in vitro. Trypsin is used to cleave proteins bonding the cultured cells to the dish, so that the cells can be suspended in fresh solution and transferred to fresh dishes. When used for this purpose, the trypsin solution must only be allowed to be in contact with the cells for a short period of time (< 60 seconds), otherwise the trypsin may damage the cells. RMPI 1640 & 10% FBS - allow for the cells to grow again Trypan Blue Stains the cells and is used as a viability dye to help to distinguish between a living and non living cells. Living cells would exclude trypan blue ( NOT BLUE color ) while non living cells would absorb trypan blue( BLUE color) Heamocytometer The haemocytometer (or haemocytometer or counting chamber) is a specimen slide which is used to determine the concentration of cells in a liquid sample. It is frequently used to determine the concentration of blood cells (hence the name “hemo-”) but also the concentration of other cells in a sample. The cover glass, which is placed on the sample, does not simply float on the liquid, but is held in place at a specified height (usually 0.1mm).Additionally, a grid is etched into the glass of the haemocytometer. This grid, an arrangement of squares of different sizes, allows for an easy counting of cells. This way it is possible to determine the number of cells in a specified volume.The fluid containing the cells must be appropriately prepared before applying it to the haemocytometer so that requires proper mixing and appropriate concentration. The concentration of the cells should neither be too high or too low. If the concentration is too high, then the cells overlap and are difficult to count. Alow concentration of only a few cells per square results in
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