Download Indirect methods of cell counting

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.