these are the techniques used in research laboratories for analysis and study of the result.

1. FLUORESCENCE
MTT ASSAY
JC-1 ASSAY
ANNEXIN PI

2. •Generation of luminescence through excitation of a molecule by ultraviolet or
visible light photons is a phenomenon termed photoluminescence, which is
divided into two categories
• fluorescence
• phosphorescence.
Fluorescence is the property of some atoms and molecules to absorb light at
a particular wavelength and to subsequently emit light of longer wavelength,
Fluorescence is a short-lived type of luminescence.
• It is a molecular phenomenon in which a substance radiates light energy
almost instantaneously upon being struck with light from another source.
• Some energy from the incident light is absorbed by the substance, meaning
that the radiated light is typically of lower energy (and thus longer wavelength)
than that of the source.

4.  Fluorescence as an imaging modality : this includes:-
• staining very specific sub-cellular components with fluorescent molecules
•For highlighting their location in the cell,
• to study potential molecular interactions at very high resolutions.
FRET : Fluorescence resonance
energy transfer.

5.  Flow Cytometry
Flow cytometry is a high-throughput application that allows for the
detection of multiple fluorochromes in a sample consisting of thousands to
millions of cells in an extremely rapid fashion.
TIRF: Total Internal Reflection Fluorescence (Microscopy).
Epifluorescence microscopy
 Quantum dots: Quantum dots are fluorescent semiconductor
nanoparticles.

6. • MTT Cell Proliferation and Viability Assay is a safe, sensitive, in vitro assay for the
measurement of cell proliferation or, when metabolic events lead to apoptosis or
necrosis, a reduction in cell viability.
• MTT is a positively charged and readily penetrates viable eukaryotic cells. The MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction
assay was the first homogeneous cell viability assay developed for a 96-well format.
• Yellow MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide, a
tetrazole) is reduced to purple formazan in the mitochondria (cytosolic compartment)
of living cells (mitochondrial reductase enzymes are active.)
•The rate of tetrazolium reduction is proportional to the rate of cell proliferation

7. The Cells are cultured in flat-bottomed, 96-well tissue
culture plates. The tetrazolium compound MTT (3-[4, 5-dimethylthiazol-2-yl]-
2, 5-diphenyltetrazolium bromide) , is reduced by metabolically active cells
to insoluble purple formazan dye crystals. Detergent is then added to the
wells, solubilizing the crystals so the absorbance can be read using a
spectrophotometer. The optimal wavelength for absorbance is 570 nm. The
data is analyzed by plotting cell number versus absorbance, allowing
quantification of changes in cell proliferation.

8. Plate cells at 1,000 to 100,000 per well.
Incubate for 6 to 24 hours.
Add 10 μL MTT Reagent.
Incubate for 2 to 4 hours until purple precipitate is visible.
Add 100 μL Detergent Reagent(sodium dodecyl sulfate
in diluted hydrochloric acid).
Leave at room temperature in the dark for 2 hours.
Record absorbance at 570 nm.

9. These assays are used for measuring the results of cell proliferation,
testing for cytotoxic effects of compounds, and for multiplexing as an
internal control to determine viable cell number during other cell-based
assays.
 Drug sensitivity.
Cell-based assays are often used for screening collections of
compounds to determine if the test molecules have effects on cell
proliferation or show direct cytotoxic effects that eventually lead to cell
death.
 Response to growth factors.
When the amount of purple formazan produced by cells treated with an
agent is compared with the amount of formazan produced by untreated
control cells, the effectiveness of the agent in causing death of cells can
be deduced.

10. • Propidium iodide (PI) is widely used in conjunction with Annexin V to
determine if cells are viable, apoptotic, or necrotic through differences in
plasma membrane integrity and permeability.
•In apoptotic and necrotic cells, the integrity of the plasma and nuclear
membranes decreases, allowing PI to pass through the membranes,
intercalate into nucleic acids, and display red fluorescence .
•At the onset of apoptosis, phosphatidylserine is translocated to the
external membrane and serves as a recognition signal for
phagocytes.

11. An early event in apoptosis is the flipping of phosphatidylserine of
the plasma membrane from the inside surface to the outside surface. Annexin V binds
specifically to phosphatidylserine and labelled Annexin V can be used detect apoptotic
cells. Binding of Annexin V to the exposed charged head groups of PS is a Ca2+
dependent process. Propidium Iodide is used in conjunction with labelled Annexin V.
The cell membrane integrity excludes Propidium Iodide in viable and apoptotic cells,
whereas necrotic cells are permeable to Propidium Iodide.Bound to nucleic acids the
fluorescence excitation maximum of PI is at 535 nm and its emission maximum at 617
nm.

13. Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate
Assessment of Cell Death
Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

14. It is a novel cationic carbocyanine dye that accumulates in mitochondria. It exists
as a monomer and yields green fluorescence. It acts as a marker of mitochondrial
activity, thus forms J-aggregates that exhibit a broad excitation spectrum and an
emission maximum at ~590 nm which give red emission, which is reversible. These
characteristics make JC-1 a sensitive marker for mitochondrial membrane potential.
Chemical name: 5,6-Dichloro-2-[(E)-3-(5,6-dichloro-1,3-diethyl-1,3-dihydro-2H-
benzimidazol-2-ylidene)-1-prop-1-enyl]-1,3-diethyl-1H-benzimidazolium iodide .

15. JC-1 is capable of entering selectively into mitochondria,
in healthy cells with high mitochondrial (ΔψM), JC-1 spontaneously forms
complexes known as J-aggregates with intense red fluorescence. On the other
hand, in apoptotic or unhealthy cells with low (ΔψM), JC-1 remains in the
monomeric form, which shows only green fluorescence. This property is due to the
reversible formation of JC-1 aggregates upon membrane polarization that causes
shifts in emitted light from 530 nm (i.e., emission of JC-1 monomeric form) to 590
nm (i.e., emission of J-aggregate) . The main advantage of the use of JC-1 is that it
can be both qualitative, considering the shift from green to red fluorescence
emission, and quantitative, considering the pure fluorescence intensity.

16. Harvest cells (at least 2x105) from experimental samples, bring total volume up to 1
mL of fresh complete medium.
Stain cell suspension with 2.5 mg/mL JC-1. Shake cell suspension until the dye is
well dissolved, giving a uniform red-violet color.
vortex vigorously the suspension immediately after the addition of the probe.
Keep the samples in a dark place at room temperature for 15-20 minutes.
Wash twice
centrifuging at 500 g for 5 min with a double volume of PBS.
Resuspend in 0.3 mL of PBS, then analyze immediatly with the flow cytometer.

17. A distinctive feature of the early stages of programmed cell death is the disruption
of active mitochondria. This mitochondrial disruption includes changes in the
membrane potential and alterations to the oxidation–reduction potential of the
mitochondria.