ISOLATION OF HUMAN FETAL LIVER STEM CELLS WITH ALFA FETO PROTEIN, CK18 & ALBUMIN Presented by S. EZHIL MANGAI
UNDER THE GUIDANCE OF
INTERNAL GUIDE : L.G. MOHANA DIVYA Lecturer Department Of Biotechnology KSR College Of Technology EXTERNAL GUIDE : DR. ALEEM KHAN Centre For Liver Research And Diagnostics Hyderabad.
Stem cells are primal cells common to all multicellular organisms that retain the ability to renew themselves through cell division and can be differentiated into wide range of specialized cell types.
There can be two sources of stem cells- Autologous and Allogenic.
Autologous embryonic stem cells generated through therapeutic cloning and highly plastic stem cells from the umbilical cord blood or bone marrow from candidates.
Allogenic stem cells can be derived from marrow, peripheral blood, cord blood, family donors or HLA typed donors .
To isolate the fetal liver Cells from the fetus by two step collagenase method.
Separation of CD34+ cells by magnetic activated cell sorting method.
Characterization of isolated cells by polymerase chain reaction by using cell markers namely ALB, AFP, CK18.
The cell viability test was performed in total cells and in MACS sorted cells by dye exclusion method.
Trypan blue is a vital stain used to selectively colour dead tissues or cells blue. It is a diazo dye
The dead cells intake dye which results in blue colour. Whereas the viable cells remain colorless.
To characterize the isolated stem cells.
It visualizes the sub cellular localization of antigen in isolated stem cell populations .
Immunocytochemistry refers to a process of localizing proteins in the cells exploiting the binding of antibodies to antigen.
The visual marker may be a fluorophore (like FITC, rhodamine), colloidal metal, hapten, radioactive marker or more commonly for light microscopy an enzyme. Ideally, maximal signal strength along with minimal background or non-specific staining is required to give optimal antigen demonstration.
Take 5 million cells and add 10 µl of diluted primary antibody
↓ Incubate for 2-3 hrs
↓ Wash the cells by applying centrifugation at 1000 rpm for 2 mins at 4°c
Remove the supernatant and add 10 µl of diluted secondary antibody ↓ Incubate for 1-2 hrs
prepare cell smear on slide and fix using formaldehyde
↓ Observe the cells under fluorescent microscopy
MTT assay is a laboratory test and a standard colorimetric assay (an assay which measures changes in color) for measuring cellular proliferation ( cell growth).
It can also be used to determine cytotoxicity of potential medicinal agents and other toxic materials .
Yellow MTT (3-(4,5- Di methyl thiazol -2-yl)-2,5-di phenyl tetrazolium bromide, a tetrazole ) is reduced to purple formazan in the mitochondria of living cells.
A solubilization solution (usually either dimethyl sulfoxide , an acidified ethanol solution, or a solution of the detergent sodium dodecyl sulfate in dilute hydrochloric acid ) is added to dissolve the insoluble purple formazan product into a colored solution.
The absorbance of this colored solution can be quantified by measuring at a certain wavelength (usually between 500 and 600 nm) by a spectrophotometer . The absorption maximum is dependent on the solvent employed.
1.5 ml sample is added to 1 ml of MTT reagent ↓
Incubation for 2-3 hrs at room temperature
Add 1 ml of isopropanol
Vortex the sample
centrifuge at 3000 rpm for 4 mins
collect the supernatant and calculate OD at 570 nm
↓ With the readings plot a graph for absorbance vs. activity of cells.
Number of cells in small square =30
Number of viable cells =14
Number of dead cells =16
Number of cells in =30*16 large square =480
Total number of cells =480*4
Number of viable cells in =14*16
large square =224
Total number of viable cells =224*4
Percentage of viable cells
= total no. of viable cells * 100
total no. of cells
= 896 * 100
MTT ASSAY Absorbance of MTT Formazan, the Standard Procedure to Access Cell Activity: 0.9945 250 1.1436 300 0.8398 200 0.6240 150 0.4421 100 0.2621 50 0.0000 Blank OD at 580 nm Concentration of cell activity (µg/ml)
Estimation of cell activity by MTT assay: 0.1790 39 0.1349 35 OD at 540 nm Percentage of activity (µg/ml)