dr siti suri lecture 9 quantification

8/14/2019 Dr Siti Suri Lecture 9 Quantification

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QUANTITATION is required in cell culture for thecharacterization of the growth properties of different cell

lines.

Counting technology:

Hemocytometer using a slide chamber and a microscope.

2)Electronic counting by electrical resistance.

3)Stained monolayers Crystal violet, Coomasie blue,sulforhodomine B, MTT and measure the absorbance value.


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QUANTIFICATION OF CELL PROLIFERATIONCell weight ie 2.5 x108 cells /gram HeLa cells (14-16um indiameter)DNA content; assay DNA uses fluorescence method. Readfluorescence emission at 492nm.Protein; total cellular material can be measure by absorbanceof 280nm and colourmetric assays (Bradford reaction,

coomassie blue).Rates of synthesis; DNA synthesis or RNA synthesis

Estimation of DNA synthesis by (3H)Thymidineincorporation

Estimation of protein synthesis by (3H)leucine or (35S)methionine and measure with scintillation counting


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Enzymes assays and immunoassay;

Cytometry:

in situ labeling; uses fluorescence dye or conjugatedantibody to detect antigen can measure amounts of

enzymes, DNA, RNA protein, and other constituents insitu using CCD camera.

flow cytometry; of a suspension cells able to measuremultiples constituents and activities


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CELL proliferation

Measurement of cell proliferation rates are used toset conditions for routine maintenance (subculture,optimum dilution, estimate plating efficiency) or toassay differences in growth maintenance (medium,serum, culture flask etc).


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Growth cycle parameters:

Lag phase (cell adaptation and attaches to substrate, increaseenzymes activity follow by synthesis of new DNA and structuralproteins)Log phase (exponential growth (90-100%): consistent, uniform andhighest yield and reproducibility)

Stationary Phase (plateau: reduce growth fraction (0-10%),different morphology, more differentiated, secrete moreextracellular matrix and difficult to disaggregate), cellconcentration cells/ml medium in plateau.Saturation densityDuration of Population doubling time (PTD). Time taken for the

culture to increase twofold in the middle of the exponential phase. ierapidly growing cells PTD of 12-24hr, slow growing cells PTD >24hour.


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Growth Curve with a monolayers inflask

Growth Curve with suspension cultures


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Growth Curve


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Saturation Density


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Plating efficiency

Define as colony formation at low density.

Prefer method for analyzing cell proliferation and survival.

Plating efficiency=No.of colonies formed/no.of

cells seeded x100

Seeding efficiency=No. of cells attached/no. ofcells seeded x100


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Labelling Index

Cells that are synthesizing DNA will incorporate (3H)TdRIs.

Labelling Index is a percent of labeled cells as determined byautoradiography. Able to show differences betweenexponential growing cells and cells in plateau phase.

Growth Fraction; proportion of cells in cycle at time oflabeling.Mitotic Index; percentage of cells in mitosis following stainingthe cultures

Division index; percentage of cells in division cycle. Usesantibodies directed against DNA polymerase and theproportion of stained cells are determined by flow cytometry.More sensitive than DNA labeling and mitotic index.

dr siti suri lecture 9 quantification

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