Transcript rough guide
Indirect methods of cell counting Need for cell no. estimation • Consistency of primary cultures. • Maintenance of cell lines • Various other experimental analysis Direct methods 1. 2. 3. •Haemocytometer •Microscopic assays •Cell counter Indirect methods Indirect Methods :DNA estimation Protein estimation Cell weight Glucose Uptake Rough estimation 1.) Crystal violet & Densitometer. 2.)Method has also been used to calculate the number of cells per colony in clonal growth assays. 3.)Staining cells with Coomassie blue, sulforhodamine & standard plot of absorption. Cell weight • Wet weight - Amount of adherent extracellular liquid gives a large error. • As a rough guide, however, there are about 2.5 × 108 HeLa cells (14–16 μm in diameter) per gram wet weight. • Dry weight - An estimate of dry weight can be derived by interferometry. DNA CONTENT DNA Content Assayed by Several fluoroscence methods and flow cytometers. DNA dyes can bind to DNA allowing quantification of the amt. of DNA by FLOW CYTOMETRY. • Hoechst 33258 – Eg. Of fl. Dye commonly used. • This method gives a sensitivity of 10 ng/mL, • DNA can also be measured by its : Absorbance at 260 nm, where 50 μg/mL has an optical density (O.D.) of 1.0. Because of interference from other cellular constituents, the direct absorbance method is useful only for purified DNA. Flow cytometry • Use to analyse phys. And chem. Characteristics of particles in a fluid • Flouoroscently labelled cell components excites by laser to emit light at diff. wavelengths. • Flour. Can be measured to determine various properties. Protein CONTENT Protein content • Used for – I. Estimating total cellular material. II. Growth experiments. III. Expression for specific enzymes estimation. IV. Intracellular metabolite concentrations estimation. Protein can be measured by Colorimetric assays. Measuring the absorbance at 280 nm. ( Bradford & lowrys method) The absorbance at 280 nm can detect down to 100 μg of protein, or about 2 × 10^5 cells. GLUCOSE UPTAKE Glucose Uptake Used for immobalised cells Cell growth can be measured by changes in glucose levels Analyzed by measuring the rate of uptake of radioactively tagged 2-deoxy glucose. Measured by using liquid scintillation counter. SUMMARY Indirect methods rely upon the measurement of intracellular cell components such as DNA or proteins The extracellular changes such as nutrient depletion or the enzyme released by the cell can also be used for indirect cell measurment.