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ANALYSIS OF MONOCLONAL ANTIBODY STAINING OF A HUMAN CANCER CELL LINES FOR ANALYSIS USING FLOW CYTOMETRY

Introduction:

Cell culture is a procedure in which living cells are removed from a living organism and cultivated in another synthetic environment in the presence of their growth elements (Yadav, n.d). In the normal course, Analysis of monoclonal antibody of a human cancer cells undergo only a limited number of divisions before becoming unable to proliferate further.

This event is determined by the cell’s genetic makeup and is known as senescence (Freshney, 2010). Transformation is an exception to the normal process of senescence, due to which continual lineages of cells are produced (Freshney, 2010). In case of certain cells, disaggregation may be required which is brought about by adding trypsin to degrade the proteins (Yadav, n.d). Aseptic measures are very important whilst dealing with cell cultures, and depend upon number of users, space, location, storage etcetera (Freshney, 2010). Analysis of monoclonal antibody of a human cancer Cell culture is an important source of producing monoclonal antibodies (Ozturk & Hu, 2005), Analysis of monoclonal antibody of a human cancer and therefore involved in this experiment.

Flow cytometry is a means of measuring cells that undergo a streamline flow crossing a certain point (Ormerod, 2000). Parameters measured include cell-size and the expression of cell-surface and intracellular markers. Analysis of monoclonal antibody of a human cancer This is why flow cytometry in biomedicine can be used to measure the fluorescence emitted by labeled antibodies, bound to individual cells in a mixed population. Analysis of monoclonal antibody of a human cancer Fluorescence-activated cell sorting (FACS) is a more sophisticated system, which quantifies the fluorescent signal and separates the cells from a mixed population that contains preselected characteristics (i.e. fluorescence intensity, size and viability), to determine the proteins that act as surface antigens in a group of cells (Chalfie & Kain, 2006).

The purpose of this report is to provide an analysis of staining of a human cancer cell with the help of a monoclonal antibody using flow cytometry.

Material and Methods:
In the laboratory, we used monoclonal antibodies to stain human cancer cell (hepatoma) lineages so as to analyse them using flow cytometry. There were three groups, each of them receiving different cell treatments as follows:
• No treatment
• VEGF (30ng/ml for 48hrs)
• VEGF (30ng/ml for 48hrs) in the presence of an anti-VEGF monoclonal antibody (120ng/ml for 48hrs
The cells were then calculated using a heamocytometer, after dilution of the cell suspension the according to the formula:

Dilution Factor (D) = Final volume after dilution ÷ Original volume of cell suspension
Thereafter, the percentage viability of cell suspension was calculated as follows:
Viability (%) = mean live cells x 100
mean total cells (alive +dead)

The number of viable cells per cm3 were then calculated from the stock cell suspension. This was done using the formula

Cells/cm3 = N x (1/V) x D

N = mean viable cell number per corner square
V = volume of liquid contained above the corner square in cm 3 ( V = 10-4, therefore 1/V = 104)
D = dilution factor due to adding trypan blue

The antibody staining of HepG2 cells with a monoclonal antibody was then performed. Included in this experiment were negative controls (without any stain) and positive controls (created by staining HepG2 cells with green fluorescent. Indirect staining was established in which the primary antibody is not fluorochrome labeled but is detected by a second fluorochrome labeled antibody. In this experiment, indirectly staining was performed using a monoclonal antibody for the protein Claudin-1. The secondary antibody used was a 488nm fluorescent cytochrome which is identifiable in the green light wavelength.

Last step performed was using flow cytometry to quantify the staining of human hepatoma cells with a monoclonal antibody for the tight junction protein Claudin-1
The staining procedure involved making a single cell suspension from cell culture or tissue samples. The cells were then incubated in tubes or microtiter plates with unlabeled or fluorescent labeled antibodies. Cells were then analyzed on the flow cytometer
Results:

Analysis of samples used in an indirect fluorescent staining protocol was done. We were provided with three samples from our experiment. These were
• Negative Control (non-stained HepG2 cells)
• Experimental sample (Claudin-1 stained HepG2 cells)
1. No treatment
2. VEGF (30ng/ml for 48hrs)
3. VEGF (30ng/ml for 48hrs) in the…