Introduction
The technique in which cells usually of one type, removed from carnal tissue are grown in presence of addendums and growing factors under controlled conditions is called Cell Culturing. Different cells require different conditions of temperature and gas for their appropriate growing in a cell brooder moreover growing media plays a important function in cell culturing, which can change in glucose concentration, pH, growing factors like foetal calf serum (FCS) .
The consequence of one of the above mentioned variables, we are traveling to analyze in this experiment is growing factors. Growth factors as the name suggests enhancers the growing which supplements the media are obtained from carnal blood such as calf serum.
Cell proliferation is studied by assorted methods, chiefly used methods affect mensurating the coloring material strength taken up by DNA of cell during cell proliferation in presence of staining dyes e.g. crystal violet and MTT ( 3- ( 4,5-dimethyl thiazol-2-yl ) -2,5-diphenyl tetrazolium bromide ) .
The purpose of this experiment is to analyze cell proliferation by utilizing crystal violet and MTT methods while utilizing mouse Y1 adrenocortical cells under the consequence of growing factor FCS.
Mouse Y1 adrenocortical cell are grown in Dulbecco ‘s alteration of Eagle ‘s medium as monolayer civilizations.
Crystal Violet
It is biological discoloration used in cell proliferation method which stains Deoxyribonucleic acid of the cells after permeabilisation with methyl alcohol. The positive ammonium ions of crystal violet discoloration DNA of cells in bluish coloring material.
The coloring material strength produced is straight relative to figure of cells, which is determined in stained cells by tintometer. Staining with crystal violet and MTT are based on principal of optical density.
MTT
The MTT check is a standard colorimetric check in which activity of reductase enzymes are measured which convert MTT to blue indissoluble formazan. The metabolic activity of feasible cells is used as basic parametric quantity in MTT check. The tetrazolium salt, MTT is now widely used in quantitative measuring of cell proliferation. This method depends upon consumption of MTT by feasible cells, which is a H2O soluble tetrazolium salt bring forthing a xanthous solution. MTT is cleaved to insoluble bluish formazan in the chondriosome of life cells by succinic dehydrogenases in the mitochondrial respiratory concatenation.
Material Used
Mouse Y1 adrenocortical cells, Dulbecco ‘s medicine of Eagle ‘s medium ( DMEM ) incorporating 10 % Fetal calf serum, 2mM glutamine, 100u/ml penicillin and 100mg/ml streptomycin, crystal violet discoloration ( 0.1 % solution in 200mM boracic acid ) , MTT ( 5mg/ml MTT solution in PBS ), PBS methyl alcohol, 10 % glacial acetic acid, distilled H2O, acid-isopropanol.
Apparatus Used
Laminar flow cabinet sterilized two 96 good home bases, multi good pipettes, grazing land pipettes, sterilized T-flasks, sterilised empty reservoirs, gas brooder, fume closet, spectrophotometer etc.
Method
Cells of mouse Y1 adrenocortical were separated from their substrate with tris in EDTA as they grow in monolayer civilizations. Then added same volume of medium and centrifuged after that figure of cells were counted on haemocytometer and diluted to concentration of 1.25 a…© 105 cells/ml and made it up to 30 milliliters.
Then cells were passaged into centre 60 Wellss of 96 good home base in extra with concentration of 0.25 a…© 10a?µ cells/ 200I?l in each well while outside Wellss of 96 good home base were filled with same sum of phosphate buffered saline ( PBS ) and allowed the cells to incubate overnight at 37 A°C temperature in humidified gas brooder. After that cells were washed with PBS three times and different Wellss of each home base were treated with different concentrations of FCS which is shown in table 1.
Therefore 12 Wellss of each home base were treated with 0, 1, 5, 10 and 20 % v/v concentration of FCS and both home bases were incubated for 72 hours.
One home base was used for crystal violet staining method and other for MTT check.
Crystal Violet Staining Method
For this method cell media was removed foremost of all from incubated home base and so cells were washed with PBS. After that were fixed with 200I?l of methyl alcohol for 15 proceedingss in fume closet. Then methyl alcohol were removed and cells were allowed to dry in fume closet for few proceedingss.
Then cells were treated for 20 proceedingss with crystal violet discoloration 200I?l/well. Subsequently cells were washed three times with distilled H2O and stained cell bed was allowed to solubilised in the 50I?l of 10 % glacial acetic acid and home bases were incubated for 30 proceedingss in gas brooder.
After that optical density of each well was read by plate reader spectrophotometer set at 540nm.
MTT Method
To execute MTT assay, each of Centre 60 Wellss of 96 good home base was treated with 20I?l of MTT solution and home base was incubated for 4 hours at 37o C temperature in gas brooder.
After 4 hours, the medium was removed from each well and 100I?l of acid-isopropand was added to fade out bluish formazan crystal in the cell bed. Then home base was incubated for 30 proceedingss at room temperature.
When bluish formazan crystal were solubilised, optical density of each well was measured at 570nm utilizing the home base reader.
Calculations
- Cells in five squares of Haemocytometer = 24
- Volume of each square is =4A-10-3I?l
- The no. of cells in five squares multiplied with 5A-104 gives no. of cells in 1ml.
- Hence no. of cell in 1ml = 1.2A-106 cells/ml
- Required cell suspension = 1.25A-105 cells/ml
- Dilution Factor = Concentration Required/Concentration got
- Dilution Factor = 0.104
- Therefore, in order to do 30 milliliter of cell suspension 3.125 milliliter of cell suspension was assorted with 26.875 milliliters of medium.
- Similarly 30 milliliter of cell suspension was prepared holding 1.25A-105 cells/ml.
Graph demoing consequence of FCS with Crystal Violet Method
Above graph shows that with addition in serum concentration the optical density additions, which is straight relative to cell figure.
Graph demoing consequence of FCS with MTT staining
Above graph shows that with addition in serum concentration the optical density additions, which is straight relative to cell figure.
Discussion
Crystal Violet Staining method and MTT Assay is based on rule of optical density, more is color strength, more will be the optical density value.
The consequence of Crystal violet staining method clearly indicated that optical density value was straight relative to cell proliferation as it was increasing with concentration of FCS. FCS stimulated Cell Proliferation Consequence in more cells and Deoxyribonucleic acid Methanol increased cell membrane permeableness Consequence in more stained Deoxyribonucleic acid More Color strength Therefore More Absorbance Value
Similar consequences were seen in MTT Assay but in this check merely feasible cells were stained while in crystal violet method both feasible and non feasible cells were stained. So Crystal Violet method of staining is non specific staining technique because in this optical density is non direct index of cell viability.
The drawback of MTT Assay is that some cut downing agent may cut down MTT besides which could demo little addition in optical density, furthermore this method depends on some variable like pH, presence of D-glucose and pyridine bases which can impact the specificity of Assay.
In malice of above said restrictions these methods are largely followed because they are safe, simple, inexpensive and consistent.
Differentiation of K562 cells to megakaryocytes/platelets
To analyze cell distinction of K652 cells chronic myelogenous leukemia, K652 cell line, indicates an early distinction phase of granulocyte line of descent. K652 cells are non-adherent, round shaped with little microvilli.
In the presence of tumour boosters like phorbol myristate ethanoate ( PMA ) these type of cell are differentiated to megakaryocytes.
The initiation of megakaryocytic distinction of K652 cells is known to be initiated by two signalling tracts which are the atomic factor – kappa B ( NF-I?B ) -depends tracts and other is extracellular signal – regulated kinase ( ERK ) /mitogen – activated kinase ( MAPK ) – dependant tracts.
Human chromic myelogenous leukemic cells, K652 cells have Philadelphia chromosome. Tumour booster, PMA which is a powerful mitogen for human peripheral blood lymph cell besides act as a protein kinase C ( PKC ) activator which differentiate K652 cells to megakaryocytes.
The assorted alterations that occurs during distinction of K652 cells are:
- Changes in cell morphology
- Cell growing apprehension
- Adhesive belongingss of cell alteration
- Expression of markers associatated with megakaryocytes
- Endomitosis
- NADPH oxidase composite which is known as a primary beginning of ROS ( Radio active O species ) , is initiated by PMA.
- PMA stimulates NADPH ROS ( Signalling Molecule )
- Initiation of cistron look is straight related with ROS.
The Expression of CD61, a thrombocyte cell marker helps in placing differentiated cells. The look of CD61 can be seen on thrombocytes, osteoclasts, macrophages and on some tumor cells, involved in tumour metastasis and in adenovirus infections.
Consequences and Discussion
It was observed that PMA treated slide was stained pink while cells devoid of PMA were stained blue as shown in Pic. 1 & A ; 2. In PMA treated slide the K562 cells were clearly differentiated to megakaryocytes which suggested that tumor booster, PMA induced distinction in K562 cells by signal transduction and expressed by CD61 as shown in image below.
Degree centigrades: UsersmkkaushalPicturescell bio picsmail2.jpg Pic. 1
PMA Treated Cells clearly demoing Differentiation to Megakaryocytes
Degree centigrades: UsersmkkaushalPicturescell bio picsmail.jpg Pic. 2
Cells Devoid of PMA stained blue in Colour
The look of CD61 was recognised by add-on of coney anti-mouse IgG antibodies that bind to CD61 antibodies when incubated in presence of alkalic phosphatise anti alkaline phosphatise ( APAAP ) composite.
The cells were stained pink because fast ruddy dye acquire attached to APAAP so this is how CD61 was expressed in cell treated with PMA. Furthermore cells treated with PMA were larger, irregular, in form and fewer in figure as comparison to untreated cells.
On the contrary, Cells devoid of PMA were much smaller in size than treated cells. Diagramatic Representations of Immunocytochemical Chemical reactions To Detect CD61 PMA Treated Cells PMA Untreated Cells CD61 edge to both treated and untreated cells Then Cells are washed to Remove CD61 unbound & A; Treated with RAM Then RAM binds to APAAP and cells are stained pink in coloring material. : Mouse Antihuman Cadmium 61 ( Primary Antibody ): Rabbit Antimouse IgG ( RAM- Secondary Antibody ): Mouse Alkaline Phosphate AntiAlkaline Phosphatase ( APAAP Tertiary Antibody )
