T he study of m ouse Y 1 aderenocortical cell proliferation using crystal violet and M T T .

1. Introduction: Cell proliferation is an key method to provide the purpose of studying important biological factors such as bioassays, toxicological tests and other carcinogenic analysis. For these analysis growth factor, serum batch testing, anti-cancer drug screening and determination of cell number is required. Reference: Animal cell culture and technology. Michael Butler.,1996, Published by Oxford University Press. Materials and Methods: Cell proliferation is a important method to study many biological activities. However for the cell proliferation, passaging is required mode to get the desired result. The two methods were used in the experiment to determine the cell proliferation is described below. Materials used for cell culture: 1) Flask. 2) Centrifuge. 3) Sterile Multi and pasture pipettes. 4) Haemocytometer. 5) Gas incubator. 6) Two 96 well plates. 7) Cell culture. Cell culture Methods: The passaging of cells in which the subculture should be removed from the monolayer culture substratum by using enzyme trypsin (0.05% or EDTA (0.02%). The passage cells should be transfer into the 60 wells in the centre of the 96 well plates by maintaining a density of 0.25% × 105 cells/well (200µl of 1.25×105 cells/ml in the cell suspension). Fill up the wells with 200µl of phosphate buffered saline (PBS). Allow them to settle for a night in gas incubator. Put the cell incubations in two 96 well plates. Next cells was treated with medium containing 0,1,5,10 and 20% v/v serum which is in 12 wells. Medium should be dilated containing serum and required 3ml of each in treatment per 96 well plates ( 200µl/well). The cell should be washed 3 times with PBS by using multichannel pipette and media containing different serum should be added. The two different plates where as one should be used for crystal violet uptake and another for MTT assay it was putted for 48 hours incubation. 1 8) Trypsin/EDTA (TE). 9) Dulbecco's modification of Eagle's medium (DMEM). 10) Phosphate Buffer saline (PBS).

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A) Staining with Crystal Violet: This method is based on the principle of estimating cell proliferation by calculating the absorbance after staining the culture cells with crystal violet which is taken up by the viable cells in medium with different concentration.

Material Used: 96 well plates for cell culture. Pasture and multichannel Pipette. Gas incubator. Crystal Violet. Methanol. Foetal calf serum (FCS). Phosphate Buffered Saline (PBS). Medium. Glacial Acetic Acid. The cell cultures from the 60 central wells of the 96 well plate incubated earlier were removed and the wells were washed 3 times with PBS using Pasteur pipette. Medium with serum, 5 different concentrations were prepared in the following composition and was incorporated into those 60 central wells of the plate (12 wells Per treatment) and was incubated for 48 hours.
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After 48 hours incubation cell cultures from the 60 central well of the 96 wells plates were removed and the wells were washed with 200µl PBS. Cells were then fixed with 200µl methanol for 15 minutes. Then methanol was removed and the plates were removed and the plates were allowed to dry in the air in the fume cupboard. After drying the plates were stained with 200µl crystal violet per well for 20 minutes. The plates were then washed three times with distilled water and the stained cell layer was solubilised in 50µl glacial acetic acid. The plate was incubated for 30 minutes in gas incubator to achieve the dissolution. The absorbance of the wells were later read using plate reader at 540nm. The absorbance of the cells of different concentration were taken and their mean was calculated. A graph was plotted using the resulted mean.

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Table: 1 Composition of different serum concentration in the media: S.No. 1 2 3 4 5 Concentration of FCS 0% 1% 5% 10% 20% Mean O.D. at 540nm 0.618 0.789 0.964 1.047 1.385

Serum Conc. vs Avg OD Crystal violet method
Avg OD @ 540nm
1.5 1.4 1.3 1.2 1.1 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 1 5 10 20

Serum Concentration

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Result: It can be noted from the graph that it is a continuous increase of cell proliferation in the presence of serum upto 20%. It was noted that the colour of cells appeared as pink after the experiment.

B) MTT method: MTT should be added 20µl (5 mg/ml solution in PBS) to each of the treated well of the 96 plate for 4 hours. Then it was kept again the 96 well plate for 4 hours into the gas incubator at 37°c. After 4 hour the medium was removed by using pasture pipette. The 100µl of acidisopropanol was added to each well to dissolve the formazan crystal in the cell layer. Then it was incubated at room temperature with the lid of the plate on for 30 minutes. Blue formazan crystal was solubilised and the absorbance of the each well was taken at 570 nm by using plate reader. 3

Table 2: Composition of different serum concentration in the media.

S.No. 1 2 3 4 5

Concentration of FCS 0% 1% 5% 10% 20%

Mean O.D. at 570nm 0.126 0.157 0.240 0.311 0.458

Serum Concentration vs Avg OD MTT method
Avg OD @ 570nm

0.5 0.4 0.3 0.2 0.1 0 0 1 5 10 20 Serum Concentration

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Results: It can be noted that rate of proliferation was increased after 1.5% of serum concentration. The maximum growth was attained around 20%. It was observed that the cells appeared in sky blue colour because of the addition of formazan crystal. Discussion: It was noticed that there is no decrease rate of cell proliferation in the experiments. With this it can be interpreted that the increase in the concentration of FCS increased proliferation to an extent that can be considered as optimum concentration beyond which increase of concentration may give a negative or no effect on the cell proliferation.

2. Two other methods used in the determination of cell proliferation. There are various methods to determine the cell proliferation which has become a common practice in scientific laboratories. However the method should be easy, economical as well as safe.

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a) almarBlueTM Reduction: A new method for measuring cell proliferation has in recent times become accessible which uses almarBlue TM. As with tetrazolium salts, almarBlueTM monitors the dropping environment of the proliferating cells. AlmaeBlueTM is soluble, stable in culture medium and is non-toxic. The continuous monitoring of cells in culture is therefore permitted. Specifically, almarBlueTM does not alter the viability of cells cultured for various times as monitored by Trypan Blue exclusion. Cells grown in the presence of almarBlueTM and subsequently analysed by flow cytomeytry for CD44, CD45RB, CD4, and heat stable antigen are found to produce similar numbers of viable cells and antigen expressing cells as non-almarBlueTM exposed cells. On the other hand almarBlueTM does not interfere with the ability of hybridomas to secrete antibody. Because almarBlueTM is non-toxic, the cells under study can be returned to culture or used for other purposes including histological studies. Proliferation measurements with almarBlueTM may be made of eigther spectromhotometrically by monitoring the absorption of almarBlueTM supplimented cell culture media at two wavelengths, or alternatively, proliferation measurement with almarblue may be made flurometrically. Proliferation may therefore be monitored with almarblue using a standard spectrophotometer or by spectroflurometer. Spectrophotometric microtitric well plate reader and spectroflurometric microtiter well plate reader could be used. b) Neutral Red uptake method: The quantitation of Neutral Red also has utility in measuring cell proliferation. Proliferation assays are performed by culturing cells in micrititer well plates or trays followed by neutral Red staining. The cells under study may be in suspension or adherent. The absorbance of the Neutral Red stained cells monitored at 550 nm is indicative of the cell number. Results obtained by this method in the measurement of cells proliferation in response to the cytokines IL-2 and IL-6 compare favourably with results obtained by measuring 3H-thymidine incorporation . Reference: http://www.biosource.com/content/literatureContent/PDFs/alamarbluebooklet.pdf

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Immunocytochemistry:
1) Cell Differiention of K562 cells to megakaryocytes: The K562 ( Chronic myelogenous leukemia cell line) has been comprehensively used in the studies of cell differentiation. Phorbol Myristate acetate (PMA), which can also function as a tumour promoter, was used in the experiment to differiention of K562 cells to megakaryocytes. The differiention process was monitored by the expression of platelet cell marker and also vitronectin receptor-CD61. 5

Method: All were incubated in presence and in absence of PMA, two slides were prepare by cytospin. Both the slides were then treated with mouse anti-human CD61. Both the slides were then treated with rabbit anti mouse IgG and Alkaline phosphatase anti-alkaline phosphatase complex ( APAAP ) respectively. Incubating and washing was done in between each treatment. The slides were then treated with fast red TR substrate. The slides were washed and then counter stained with haematoxylin. It was then washed with TBS and mounted in Glycerol/TBS and viewed under microscope.

Results: It was noticed that cells which were incubated with PMA and treated with CD61 produced pink colour. When cells treated devoid of PMA did not show pink. It indicates the cell proliferation. In the presence of PMA, CD61 inhibits the cells. This can be recognised by the addition RAM and APAAP,RAM attaches to CD61 and APAAP attaches to RAM. This can be confirmed as the cells with complex only stained pink. Reference: Immunochemical Methods and Protocols. Lorette C. Javois., 2nd ed, 1999. Published by Humana Press Inc. Immunochemistry 2. A Practical Approach. A.P.Johnstone and M.W.Turner., Published by Oxford University Press. 2) Agents used to induce differentiation in cell culture: • Sodium butyrate • Hemin • Butyric acid • Retinoic acid. .Cisplatin etc.
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The cell line K562 is a human erythrolekemia which can be induced to erythroid terminal differentiation by the alkylting agent as Cisplatin. Biologists demonstrated that it could be used for the treatment for solid cancer. These cell produces haemoglobin. In the process of differentiation at the early stage, can down the regulation of transferrin receptor which is highly expressed on the cell surface. moreover the Tf receptor activity is related with the cell growth which could be down regulated and lose terminal differentiation. From this could be noted that CDDP can induce the terminal differentiation in K562 cells. Reference: Cisplatin-induced erythroid differentiation in K562 cells: modulation of transferrin receptor.Parodi MT, Tonini GP, Bologna R, Franchini E, Cornaglia-Ferraris P. Boll Ist Sieroter Milan. 1988;67(2):142-8. 3. The effects of PMA on intracellular signalling pathways PMA is a common practice which is used to stimulate NADPH oxidase activity in the cells. NADPH oxidase complex is recognised as a primary source of ROS (Radio active oxygen species). 6

ROS are the species which are in a more reactive state than molecular oxygen and therefore is reduced to varying degrees. The primary ROS is superdioxide, which is formed by the reduction of the one-electron of molecular oxygen. This reaction is catalysed by NADPH oxidase. For the further reduction of oxygen, produces hydrogen peroxide. Thereafter further reaction may lead to the formation hydroxyl radicals, in the presence of metal ions through the Fenton reaction. Hydroxyl radicals are extremely reactive, with a short half life and react with the first molecule. In neutrophils, myeloperoxide catalyses the formation of hypochlorous acid while superoxide may also react with nitric oxide to form another relatively reactive molecule as peroxynitrite.The formation of superoxide anions, cascade of ROS production is likely. ROS especially hydrogen peroxide are the main signalling molecule. ROS are generally useful as signalling molecules and in animal as well as plant host defence. However on the other hand they might produce cellular damage if they produce in an uncontrolled manner.

PMA

stimulates

NADPH oxidase

ROS (signalling molecule)

ROS has also some direct effects as induction of gene expression and involvement of phosphorylation. It also alter the redox status of the cell. Reference: Intracellular Reactive Oxygen Species Mediate the Linkage of Na /K -ATPase to Hypertrophy and Its Marker Genes in Cardiac Myocytes.,Zijian Xie‡, Peter Kometiani, Jiang Liu, Jie Li, Joseph I. Shapiro, and Amir Askari. Vol. 274, No. 27, Issue of July 2, pp. 19323–19328, 1999

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