Importance Of Cell Culture And Cell Counting

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Cell culture & cell counting:
Introduction: Cell culture is the method of removal of cells from their respective animal or plant source, followed by their growth in a favourable artificial environment. These cells can be removed directly from tissues or fragmented using enzymatic or mechanical method. These cells can be obtained from the already established cell line or cell strain.
Short protocol:In this protocol, initially cells were washed to remove adherent cells, incubated and supernatant was removed. Pellet was examined, resuspended in the fresh medium and incubated in the cell culture medium. For counting cells were washed to remove adherent cell, centrifuged, resuspended in required medium and counted using haemocytometer.
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FACS performs the function of sorting of complex mixture of biological cells in different containers, depends on the respective fluorescent and light scattering properties of cells in the mixture of cells. In FACS, per second, thousands of individual cells can pass through the laser beam and these cells get separated depends on the properties of the cells i.e. size, healthiness, heterogamous nature, and phenotype. FACS is useful for the quantitative separation of the cell of interest and also to separate the subpopulation of the cells.
Short protocol:Broadly, in this protocol initially FACS/Sort medium was prepared and made different aliquots, followed by preparation of single cell suspension and separation of big, granular and small cells and cells were checked daily and medium each 4-5 days. After sorting, larger cells were used for further culture and cloning.
Use of substances:In this protocol FBS was used as serum supplement which contains growth factors. HEPES was used to provide buffering capacity to the cell culture medium. RPMI was used as medium for cell culture. Gentamycin and Ciprofloxacinwere used to prevent the contamination of the culture and
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In this technique, antibody linked to an enzyme is complexed with the immobilized antigen.Conjugatedenzyme activity is measured by incubation with substrate.
Short protocol:Briefly ELISA procedure describes: antibody was coated on the plate, incubated, washed and blocked with BSA. Again plate was washed, sample and IL-6 was added, incubated, washed and detection antibody was added, incubated and washed. Avidin- HRP was added, incubated and washed. SuperAqua Blue solution was added and absorbance was measured.
Use of substances:In ELISA, bovine serum albumin (BSA) was used as a blocking agent to avoid non-specific binding of antigens and antibodies to the microtiter wells. Washing buffer was used to remove, the coating material. In ELISA, Avidin–horse-radish peroxidase (HRP) solution was used. Avidinhas high affinity with biotin and binds with it. Thus, avidin-HRP is valuable for detecting biotinylated antibodies. Super AquaBlue provides high signal, high sensitivity, and low background. HRP oxidises super AquaBlue ELISA and produces soluble blue-green colored end product. This end product can be measured at 405

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