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Cell viability could be referred as the ability of cell to survive. Number of cells present in tissue could be determined by introducing dyes into the cells in the presence of proper light. This method has its important significance in the field of Biology. There are many fluorescent which are used in laboratories; Calcein is a fluorescent dye which is used widely today. Calcein analysis helps in the effectiveness of chemotaxis, multidrug resistance and cell adhesion. Calcein AM is a derivative of acetoxymethyl ester which has an ability to be transported into live cells via cell membrane. This fluorescent is helpful to test the cell viability. Membrane of living cells needs to be in tact and calcein works in living tissue. One needs to remember that calcein works only for living cells, if the cells are dead, then Calcein would be of no use.
The chemical formula for Calcein is given below:
Purpose of Experiment
The main purpose of this experiment was to identify the number of cells present in a tissue. This experiment showed the use of different chemicals such as Calcein, PBS, and RPMI media.
Material which I used in the process of identifying the number of cells is as follows:
PBS ( Phosphate Buffered Saline)
RAW 264.7 cells
CytoFluor II fluorescent plate reader
The media, I used for this experiment was RPMI which was in a liquid form with sodium bicarbonate and L-glutamine and sterile-filtered. First of all, I took some wells with some cell tissues for the experiment. Wells were kept in 4 rows. Each row had 4 wells. Each of the well was added with 0.5 ml of Phosphate Buffered Saline. All the wells were kept for some minutes with BPS. After one or two minutes, PBS was poured out from wells. Well were left for some time. 0.5 ml of PBS was again added into each well. Wells with PBS were again kept for one minute. The use of Calcein started after staying PBS. Calcein was not added in the first four wells, rests of the wells were added with Calcein. I kept all these mixtures warm for 15 minutes. Fluorescent plate reader CytoFluor II was used to determine the fluorescent of cells. The fluorescent plate was provided with media RPMI and fluorescent plate reader read the quantities of cells presented in wells.
The result of the experiment shows that the first row of 4 wells which was not added with Calcein did not show any reaction but the wells which were added with Calcein increased the number of cells.
Number of cells presented in each row is given below:
Number of cells in first row was : 1.5* 104
Number of cells in second row was : 2.5 *105
Number of cells in third row was : 7.5*105
The following Bar graph shows the graphical represented of data generated from fluorescent plate reader. In this experiment I used 4 rows of wells to find the number of cells. The first row did not show any increment in the number of cells due to the absence of Calcein. The three rows which showed increment in the number of cells are plotted in Bar graph. Readings were repeated 4 times which are shown in following graph.
The graphical representation of Value of Calcein v/s number of cells is shown below.
Value of Calcein
No. of cells
The plate was into the CytoFluor II reader, readings from all the concentrated wells were different. The graph shows the average fluorescent units for each of the row of wells at the same concentration.
The cells were washed with Phosphate Buffer Saline (PBS). BPS was used to clean some extra molecules present in the extra cellular solution. The fluorescent dye, Calcein which I used in this experiment helped the enzymes to work for the growth of cells. Neutral or near-neutral molecules easily diffuse with most of the cells. After reaching into the cell medium, it starts loading of acetoxymethyl ester. After the conversion of acetoxymethyl ester, inflorescent substrates are converted into intracellular esterase that is kept by cells within the plasma membrane. The complete experiment shows the use of Calcein in cell culture. Different type of Calcein's is used in different biological activities but the use of Calcein AM has its special significance in identifying the number of cells present in tissue.
Assay cell viability & live-cell function, Retrieved February 14, 2008, from http://probes.invitrogen.com/media/publications/442.pdf
Advancing Life Sciences Research, (2007), Retrieved February 14, 2008, from http://www.moleculardevices.com/?gclid=COfE15zfw5ECFQx0bgodFyJZDA
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