Cell Proliferation Assay
|Affiliation: * Shalini Jain and Hariom Yadav|
Animal Biochemistry Division, National Dairy Research Institute, Karnal-132001, Haryana, India
|Date Added: Mon Feb 02 2009|
|Date Modified: Mon Feb 02 2009|
Living (metabolically active) cells reduce tetrazolium salts to colored formazan compounds; dead cells do not. Thus, tetrazolium salt-based colorimetric assays detect viable cells exclusively. Because they are sensitive, these assays can readily be performed in a microplate with relatively few cells. Since a cytotoxic factor will reduce the rate of tetrazolium salt cleavage by a population of cells, these metabolic activity assays are frequently used to measure factor-induced cytotoxicity or cell necrosis. Applications include:
Measurement of cell viability and proliferation forms the
basis for numerous in vitro assays of a cell population’s response to external
factors. The reduction of tetrazolium salts is now widely accepted as a reliable
way to examine cell proliferation. MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, first
described by Mosmann in 1983, is based on the ability of a mitochondrial
dehydrogenase enzyme (by generating reducing equivalents such as NADH and NADPH)
from viable cells to cleave the tetrazolium rings of the pale yellow MTT and
form a dark blue formazan crystals, which is largely impermeable to cell
membranes, thus resulting in its accumulation within healthy cells. The
resulting intracellular purple formazan can be solubilized and quantified by
spectrophotometric means. Solubilisation of the cells by the addition of a
detergent results in the liberation of the crystals which are solubilized. The
color can then be quantified using a simple colorimetric assay. The results can
be read on a multiwell scanning spectrophotometer (ELISA reader). The MTT Cell
Proliferation Assay measures the cell proliferation rate and conversely, when
metabolic events lead to apoptosis or necrosis, the reduction in cell viability.
Here we took splenocytes for cell proliferation assay.
Preparation of basal culture medium (RPMI-1640)
The following ingredients were added to one litre of
autoclaved and cooled triple distilled water:
RPMI 1640 16.4 g
NaHCO3 2.10 g
Sodium pyruvate 110.1 mg
HEPES 5.96 g (50 mM)
L-Glutamine 293 mg
Pencillin 61 mg (100 IU/ mL)
Streptomycin 100 mg (100 μg/ mL)
Adjust the pH of the resulting solution to 7.2 with the help
of 1N NaOH and sterilize through 0.22 μm Millex-GV filter unit (Millipore).
Store the prepared medium in small aliquots at 5°C in
Erythrocyte lysis buffer
10 ml of 0.17M Tris
90 mL of 0.6M of NH4Cl
MTT was dissolved in RPMI-1640 at 5 mg/ mL and filter through 0.22 μm filters.
Preparation of culture medium
Supplement the basal culture medium with 10% fetal calf serum
(FCS-heat inactivated at 560 C for 30 min) for its use as culture medium and add
mitogen i.e., Lipopolysaccharide (LPS; 10-50 μg/ mL) to proliferate the cells.
Preparation of splenocytes
Dispensing and culturing of lymphocytes:
Cell proliferation assay
A540 nm with LPS
Stimulation index = -------------------------------
A540 nm without LPS
Where A 540 = Absorbance at 540 nm.
Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to
proliferation and cytotoxicity assays. J Immunol Methods. 1983 Dec 16;65(1-2):55-63.