• Membrane integrity is the feature most often used to detect whether eukaryotic cells

cultured in vitro are alive or dead. Cells that have lost membrane integrity and allow
movement of otherwise non-permeable molecules are classified as non-viable or dead.
• Detection of dead cells is accomplished by measuring movement of molecules either
into or out of cells across membranes that have become leaky and cannot be repaired.
Indicators of dead cells include markers that exist in the cytoplasm of viable cells, but
leak into the surrounding culture medium upon loss of membrane integrity. The marker
can exist naturally such as an enzyme, or be introduced artificially, such as loading
radioactive [51Cr] or a fluorescent marker into viable cells.
• Artificially introduced markers enable selective detection of target cell cytotoxicity for
experiments using more than one population of cells such as cell mediated cytotoxicity.
A second class of molecules that serves as an indicator of dead cells is referred to as
“vital dyes”. These dyes typically are not permeable to viable cells, but can enter dead
cells through damaged membranes. Examples include trypan blue and many
fluorogenic DNA binding dyes. Addition of these molecules to cells in culture results in
selective staining of the dead cells.
• Dyes That Selectively Penetrate Dead Cell-trypan blue
• Fluorescent DNA Binding Dyes That Penetrate Dead Cells-Propidium iodide
• Markers That Leak Out of the Cytoplasm of Dead Cells into Culture
Medium-LDH
• LDH-A common method for determining cytotoxicity is based on
measuring the activity of cytoplasmic enzymes released by damaged cells.
Lactate dehydrogenase (LDH) is a stable cytoplasmic enzyme that is found
in all cells. LDH is rapidly released into the cell culture supernatant when
the plasma membrane is damaged, a key feature of cells undergoing
apoptosis, necrosis, and other forms of cellular damage. LDH activity can
be easily quantified by using the NADH produced during the conversion of
lactate to pyruvate to reduce a second compound in a coupled reaction into
a product with properties that are easily quantitated
• The sulforhodamine B (SRB) assay is used for cell density determination, based on the measurement of
cellular protein content. The method described here has been optimized for the toxicity screening of
compounds to adherent cells in a 96-well format. After an incubation period, cell monolayers are fixed with
10% (wt/vol) trichloroacetic acid and stained for 30 min, after which the excess dye is removed by washing
repeatedly with 1% (vol/vol) acetic acid. The protein-bound dye is dissolved in 10 mM Tris base solution for
OD determination at 510 nm using a microplate reader. The results are linear over a 20-fold range of cell
numbers and the sensitivity is comparable to those of fluorometric methods. The method not only allows a
large number of samples to be tested within a few days, but also requires only simple equipment and
inexpensive reagents. The SRB assay is therefore an efficient and highly cost-effective method for screening
• MTT assay is used to measure cellular metabolic activity as an indicator of cell viability, proliferation and
cytotoxicity. This colorimetric assay is based on the reduction of a yellow tetrazolium salt (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide or MTT) to purple formazan crystals by metabolically
active cells.The viable cells contain NAD(P)H-dependent oxidoreductase enzymes which reduce the MTT to
formazan. The insoluble formazan crystals are dissolved using a solubilization solution and the resulting
colored solution is quantified by measuring absorbance at 500-600 nanometers using a multi-well
spectrophotometer. The darker the solution, the greater the number of viable, metabolically active cells.
The Cell Proliferation Kit I (MTT) is an optimized MTT assay kit containing ready to use reagents, does not
need washing steps or additional reagents. It is a quantitative assay that allows rapid and convenient handling of
a high number of samples. The Cell Proliferation Kit I (MTT) can be used for multiple applications, such as,
• Quantification of cell growth and viability.
• Measurement of cell proliferation in response to growth factors, cytokines and nutrients.
• Measurement of cytotoxicity. Examples are the quantification of tumor necrosis factor-a or -b effects (see fig.
2) or macrophage induced cell death15,16 and the assessment of cytotoxic  or growth inhibiting agents such as
inhibitory antibodies.
• To study cell activation.
• The neutral red uptake(3-amino-7-dimethylamino-2-methylphenazine hydrochloride) assay provides a
quantitative estimation of the number of viable cells in a culture. It is one of the most used cytotoxicity tests
with many biomedical and environmental applications. It is based on the ability of viable cells to incorporate
and bind the supravital dye neutral red in the lysosomes. Most primary cells and cell lines from diverse origin
may be successfully used. Cells are seeded in 96-well tissue culture plates and are treated for the appropriate
period. The plates are then incubated for 2 h with a medium containing neutral red. The cells are subsequently
washed, the dye is extracted in each well and the absorbance is read using a spectrophotometer. The procedure
is cheaper and more sensitive than other cytotoxicity tests (tetrazolium salts, enzyme leakage or protein
content). Once the cells have been treated, assay can be completed in <3 h.
• The uptake of neutral red depends on the cell’s capacity to maintain pH gradients, through the production of
ATP. At physiological pH, the dye presents a net charge close to zero, enabling it to penetrate the membranes
of the cell. Inside the lysosomes, there is a proton gradient to maintain a pH lower than that of the cytoplasm.
Thus, the dye becomes charged and is retained inside the lysosomes. The method is cheaper, presents less
interference, and is more sensitive than other cytotoxicity tests (tetrazolium salts, enzyme leakage, or protein
content) The neutral red assay is more sensitive and requires less equipments than the estimation of cell death
by enzyme leakage using LDH. It also compares favorably to estimation of total cell number by assaying
protein content. The neutral red uptake assay is simpler, detecting only viable cells; however, once initiated it
must be completed immediately, as it is not possible to freeze the cells, as is done for the determination of total
protein assay. Nevertheless, this assay is compatible with the determination of total protein content because it
is possible to perform both the total protein content and the neutral red assays on the same culture, that is,
neutral red estimates can be obtained and then the protein determination can be carried out.In common to other
cell culture procedures, there are certain limitations due to the character of the compounds to be tested:
substances that are volatile, unstable or explosive in water, or with low solubility, present problems