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admin | Category: Phone Lookup Free | 11.08.2015
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Many fields of biomedical research, like cancer research, drug development and tissue engineering, require the use of living cells to perform a variety of assays. In order to successfully work with mammalian cell lines, they must be grown under controlled conditions and require their own specific growth medium. This article describes a typical workflow for subculturing an adherent cell line with detailed illustrations of all of the necessary steps. Why do I need to split my cells?Once a cell culture has been started, it cannot be grown indefinitely due to the increase in cell number, consumption of nutrients and increase in toxic metabolites which eventually results in cell death. After initially seeding the cells, growth starts with a lag phase and proceeds to a log phase, where the cells proliferate exponentially followed by a stationary phase where growth rate and death rate are equal (Fig. A first examination of the culture for fungal contamination, turbidity and particles in the medium as well as unexpected pH shifts, indicated by color change of the medium, can be done at the macroscopic level, by eye. There are many variations in mammalian cell morphology, but most mammalian cells in culture can be divided into three categories: fibroblastic cells (Chinese Hamster Ovary cells (CHO)), epithelial-cell-like (human cervix cells (HeLa)) and lymphoblast-like cells (human leukaemia cells (HL60)). Various cell lines not only differ in size and shape, they also differ in their growth behaviour. By removing calcium, cadherins which are involved in cell-cell interactions, are broken and cells separate from one another. However that makes it possible to check-in to a spot fairly far away from your actual location.
We are a bit skeptical to the concept of check-ins, but a lot of people seem to enjoy it on Facebook. Mammalian cell cultures are an essential tool in biology because they allow rapid growth and proliferation of different cell types for experimental analysis. Moreover researchers usually want to perform experiments on their cells several times, and therefore do not want to use up all of the cells at once.
After this, a closer check of the general cellular morphology and growth patterns should be examined using an inverted microscope.
In addition to this certain cell lines can have specific morphological characteristics, e.g.

They either growing adherent (fibroblastic and epithelial cells) or in suspension (lymphoblast-like cells).
Once separated from the growth surface and the surrounding cells, they can be easily separated and grown in new cell culture dishes.
4: Cells should be mainly attached to the bottom of the dish or flask and the media should be pink-orange in color. 5: Documentation of the cell status is very important to ensure uniform results across experiments. 8: Carefully wash cells up to three times with 5 ml pre-warmed PBS without calcium and magnesium to get rid of the fetal bovine serum (FBS) in residual culture media. 10: Gently rinse plate and transfer cell suspension into a 50 ml tube and spin them down for 5 min at 800 rpm.
13: Load the counting chamber with the cell suspension (~ 4 µl per counting area) by placing the pipette tip at the edge of the cover slip and gently expel the cell suspension. The Fuchs-Rosenthal counting chamber you can see here has a pattern of 16 areas of one square millimeter each bordered by triple lines.
When a cell line reaches about 80% confluence, the cells must be subcultured to ensure proper growth and health of the cells. Subculturing, or splitting the cells, produces new cultures with lower cell density than the original culture.
In the death phase, cells die due to lack of nutrients and inadequate living conditions.Fig. Most adherent cell lines grow as a single cell layer (monolayer) attached to glass or treated plastic substrates (coated with poly-lysine, fibronectin, collagen or gelatine).How can I subculture my cells?The most common method to prepare cells for subculture is by breaking the intercellular and cell-to-substrate connections with proteolytic enzymes like trypsin.
During the whole subculture process it is important to work in a contamination-free environment. Cells should be checked microscopically every day to ensure they are healthy (no contamination, few dead cells) and growing as expected.
The pH indicator phenol red turns yellow upon acidification as a result of metabolism products from the cells (or contaminations!) in the media. The Leica DMi1 can be equipped with a camera and screen which allows easy imaging and saving via remote control.Fig. To avoid over-trypsination which can damage the cells, check them every few minutes under the microscope.
Aspirate the supernatant and resuspend the cells in 10 ml fresh medium to fully remove the trypsin.Fig. By removing the medium and transferring the cells into fresh growth medium, the cells are given fresh nutrients and toxic metabolites are removed, allowing long-term maintenance of the culture.

Since the cells are attached to the bottom of the dish, they can be viewed easily from this perspective. Trypsin in combination with Ethylenediaminetetraacetic acid (EDTA) causes cells to detach from the growth surface. Examination of the cells at the beginning, during trypsination, cell counting, and after splitting is essential.
The culture medium should be changed during the log phase and cells should be passaged before they reach the stationary phase.In order to keep cells healthy and actively growing it is necessary to renew the growth medium and to subculture them at regular intervals. Observation should take place with a total magnification of 100 – 200x and with phase contrast, because most cells are difficult to observe in normal bright field illumination. During mitosis many cells round up, forming very refractile bright spheres that may float around in the medium. Trypsin cuts away the focal adhesions that anchor the cell to the culture dish and EDTA acts as a calcium chelator. As soon as cells have detached, add 5 ml culture medium to the dish to inactivate the trypsin.
To avoid counting cells twice at the edges of the area, count only those cells on the lines of two sides of a square. Change of culture medium can take place several times in the log phase dependent on the cell type. Dead cells often round up and become detached also but are usually not bright and refractile.
In this example, the cells touching the upper and left limits should be counted (thicker red lines). The best time to subculture cells is between the log phase and the stationary phase, before the cells reach confluence.Why do I need to examine my cells?It is important to examine the cell culture every day and immediately prior to subculturing to monitor cell health, check for contamination and determine when to split the cells. On the Leica DMi1 this can be done by simply moving the slider housing the condenser annulus, as shown.
In culture, they grow adherently and form a monolayer of polygonal cells after they have reached confluence. Trypan Blue selectively penetrates cell membranes of dead cells and stains them blue, but is not absorbed by living cells. Count the live and dead cells in 5 one square millimeter rectangles of the counting chamber.

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