MTT assay, SRB assay, thymidine uptake assay, dye exclusion, agar plate method

1. Presented by
ZAKIYA USMANI
M.PHARM 2ND SEM
DEPT. OF PHARMACOGNOSY

2. Introduction
 CANCER: Uncontrolled proliferation of genetically
altered cells.
 Derived from the repeated divisions of a mutant cell.
 Due to the effects of carcinogens, such as tobacco smoke,
radiation, chemicals or infectious agents.
 Cancer-promoting mutations may be acquired through
errors in DNA replication.
 Activate the cancer promoting oncogenes and/or inactivate
the tumor suppressor genes.

3. Need for novel anti cancer
 Development of multidrug resistance in patients.
 Long-term treatment with cancer drugs is also
associated with severe side effects.
 Cytotoxic drugs have the potential to be very harmful
to the body unless they are very specific to cancer
cells.
 New drugs that will be more selective for cancer cells

4. In vitro cytotoxicity studies
 In vitro cytotoxicity studies:
 Cytotoxicity assays on panel of human cancer cell
lines
 MTT-assay
 SRB- assay
 3H-thymidine uptake assay
 Fluorescence Dye exclusion tests
 Clonogenic assays
 Cell count assay

5. Contd..
Advantages:
 Reduce the usage of animals.
 Less time consuming, cost effective & easy to manage
 Able to process a larger number of compounds quickly with
minimum quantity
 Range of concentrations used are comparable to that
expected for in vivo studies
Disadvantages:
 Difficulty in Maintaining of cultures
 Show Negative results for the compounds which gets
activated after body metabolism and vice versa. Impossible
to ascertain the Pharmacokinetics

6. CELLPROPERTYANDASSAY
PROPERTY ASSAY
ENZYMATIC PROPERTY MTT ASSAY
MEMBRANE INTEGRITY DYE EXCLUSION TEST
DNA CONTENT/REPLICATION
STATUS
3H-THYMIDINE UPTAKE &
FLUORESCENT ASSAY
PROTEIN CONTENT SRB ASSAY
COLONY FORMING POTENTIAL CLONOGENIC ASSAY
CELL DIVISION CELL COUNTING ASSAY

7. MICROCULTURE TETRAZOLIUM
TEST (MTT ASSAY)
 MTT assay a quantitative colorimetric assay for
measuring cellular growth, cell survival and cell
Proliferation based on the ability of living cells.
 Yellow MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) a tetrazolium salt is reduced
to purple formazan by mitochondrial dehydrogenase of
living cells in which tetrazolium ring gets cleaved in
mitochondria .This enzyme has the tendency to convert
dye into purple colour

8. MTT Assay
TETRAZOLIUM
DYE
NADPH DEPENDENT
OXIDORUDECTASE ENZYME PRODUCE
BY MITOCHONDRIA
PURPLE COLORED
COMPOUND/FORMAZAN
This enzyme has the tendency to
convert dye into purple colour

9. MTT ASSAY
Receipt of Tissues Tissue Shipment Dosing
Rinsing
UVA Exposure and Post-Treatment
Incubation

10. Extraction in Isopropanol and Plate Reading
Transfer to MTT

11. SRB (SULPHORHADAMINE B)
ASSAY
 Measures whole protein content which is proportional
to the cell number
 SRB – a bright pink anionic protein staining dye that
binds to the basic amino acids of the cellular proteins

12. SRB ASSAY - Procedure
 Cell lines are counted, cultured & inoculated in 96
well plates.
 After incubation with different concentrations of test
compounds, the cell cultures are stained with SRB dye
 Washing with acetic acid removes the unbound dye
and the protein bounded dye is extracted using triss
base
 Optical density is determined by 96-well plate reader

13. 3H-Thymidine uptake assay
 The most reliable assays measure proliferation by directly
measuring DNA synthesis
 Radioactive 3H-thymidine is a nucleoside that get
incorporated into new strands of chromosomal DNA
during mitotic cell division
 Thymidine is replaced by uracil in RNA, so only DNA
synthesis is recorded

14. 3H-Thymidine uptake assay
 Thymidine incorporation
double with each cell division
radioactivity increases with
each generation
 A scintillation beta-counter is
used to measure the
radioactivity in DNA
recovered from the cells in
order to determine the extent
of cell division that has
occurred in response to a test
agent

15. Dye Exclusion Test
 This assay is relayed on the structural integrity
of the cells
 Dyes used : Trypan blue, Eosin, or Nigrosin
 Live cells possess intact cell membranes that
exclude the dye, whereas dead cells having lost
membrane integrity take up the dyes.

16. Dye Exclusion Test - Procedure
 Cells are incubated with different
concentrations of test compounds for 4 days
 Dead cells are stained with dye colour
 Specimen is centrifuged & collected in
microscopic slides
 Live cells – stained with hematoxylin eosin
 Tumor cell cytotoxicity is compared with
control – duck erythrocytes

17. Trypan blue dye exclusion test
Live
cell
Dead
cell

18. CLONOGENIC ASSAY
 Measures the growth inhibition.
 Important for the drugs that act by arresting the cells at
checkpoints in the cell cycle
 Procedure:
 Single cell suspensions are exposed to anticancer agents to be
tested.
 Suspensions are rinsed and plated in a semisolid medium.
 After 14 to 28 days, some cells will having undergone several
division form tumour colonies which can be quantified in a
visual or semiautomated fashion.
 No of colonies from treated cells is compared with untreated
colonies.

19. CLONOGENIC ASSAY

20. In vitro evaluation of
antioxidant

21. introduction
 Antioxidant is a molecule that inhibits the oxidation of other
molecules.
 Oxidation is a chemical reaction that transfers the electrons
from the substance to an oxidizing agent.
 These oxidation reactions can produce free radicals which
starts the chain reactions and causes damage and death to the
cell.
 Antioxidants can terminate the chain reactions by removing
the free radicals and inhibit the oxidation reactions.
 They do this by oxidizing themselves, so these are often called
as reducing agents such as thiol, ascorbic acid, polyphenols
etc…

22. In vitro methods
 DPPH scavenging method
 Nitric oxide scavenging method
 Hydrogen peroxide scavenging (H2O2) assay
 Ferric reducing-antioxidant power (FRAP) assay
 Superoxide radical scavenging activity (SOD)
 Phosphomolybdenum method
 Oxygen radical absorbance capacity (ORAC) Method
 Oxygen radical absorbance capacity (ORAC) Method
 Thiobarbituric acid (TBA) method

23. DPPH Scavenging method
 The molecule 1, 1-diphenyl-2-picrylhydrazyl (a,a-
diphenyl-bpicrylhydrazyl;DPPH) is characterized as a
stable free radical by virtue of the delocalisation of the
spare electron .
 The delocalization of electron also gives rise to the deep
violet color, characterized by an absorption band in
ethanol solution centered at about 517 nm.
 When a solution of DPPH is mixed with that of a substrate
(AH) that can donate a hydrogen atom, then this gives rise
to the reduced form with the loss of this violet color.

25. % inhibition of DPPH radical =([Abr – Aar]/Abr)
× 100
where Abr is the absorbance before reaction and
Aar is the absorbance after reaction has taken place
+AH
diphenyl-2-picrylhydrazyl
FREE RADICAL
Diphenylpicrylhydrazi
ne (non radical)

26. Hydrogen peroxide scavenging
(H2O2) assay
 The ability of plant extracts to scavenge hydrogen peroxide can be
estimated according to the method of Ruch et al. (1989).
 Preparation of hydrogen per oxide solution: A solution of
hydrogen peroxide (40 mM) is prepared in phosphate buffer (50
mM pH 7.4). The concentration of hydrogen peroxide is
determined by absorption at 230 nm using a spectrophotometer.
 Sample preparation: Extract (20–60 lg/mL) in distilled water is
added to hydrogen peroxide and absorbance at 230 nm is
determined
 Blank: after 10 min against a blank solution containing phosphate
buffer without hydrogen peroxide.

27. Contd..
 The percentage of
hydrogen peroxide
scavenging is calculated as
follows:
% scavenged(H2O2)=([Ai –
At]/ Ai)×100
 where Ai is the absorbance
of control and At is the
absorbance of test.

28. REDUCING POWER
METHOD
 This method is based on the principle of increase in the
absorbanceof the reaction mixtures. Increase in the
absorbance indicates an increase in the antioxidant activity
 In this method, antioxidant compound forms a colored
complex with potassium ferricyanide, trichloro acetic acid
and ferric chloride
 which is measured at 700 nm. Increase in absorbance of
the reaction mixture indicates the reducing power of the
sample

29. Total radical-trapping antioxidant
parameter (TRAP) method
 This method is based on the protection provided by
antioxidants on the fluorescence decay of R-phycoerythrin
(R-PE) during a controlled peroxidation reaction
 The fluorescence of R-Phycoerythrin is quenched by
ABAP (2,20-azo–bis(2-amidino- propane)hydrochloride)
as a radical generator
 This quenching reaction is measured in the presence of
antioxidants.
 The antioxidative potential is evaluated by measuring
the decay in decoloration. According to Ghiselli et al. (1995)

30. procedure
 120 lL of diluted sample is added to 2.4 mL of phosphate
buffer(pH 7.4), 375 lL of bidistilled water, 30 lL of diluted
R-PE and 75 lL of ABAP; the reaction kinetics at 38 C is
recorded for 45 min by a luminescence spectrometer
 TRAP values are calculated from the length of the lag-
phase due to the sample compared with standard.

31. Phosphomolybdenum method
 Total antioxidant capacity assay is a spectroscopic method
forthe quantitative determination of antioxidant capacity,
through the formation of phosphomolybdenum complex.
 The assay is based on the reduction of Mo (VI) to Mo (V)
by the sample analyte and subsequent formation of a green
phosphate Mo (V) complex at acidic pH
 Total antioxidant capacity can be calculated by the method
described by Prieto et al. (1999). 0

32. 0.1 mL of sample (100 lg) + 1 mL of reagent (0.6 M sulfuric acid
+ 28 mM sodium phosphate and 4 mM ammonium molybdate).
tube is incubated in a boiling water bath at 95
C for 90 min Then cool it at room
temperature
the absorbance of the aqueous solution is measured
at 695 nm against blank in UV spectrophotometer

33. IN VITRO EVALUATION OF
ANTIMICROBIAL

34. In vitro methode
 Diffusion methods
 Thin layer chromatography
 Dilution method
 Time kill test
 ATP bioluminescence

35. Agar disk-diffusion method
 Agar disk-diffusion testing developed in 1940
Principle
 A paper disk with a defined amount of antibiotic is
used to generate a dynamically changing gradient
of antibiotic concentrations in the agar in the
vicinity of the disk.

36. procedure
 Agar plates are inoculated with a standardized inoculum
of the test microorganism.
 Then, filter paper discs (about 6 mm in diameter),
containing the test compound at a desired concentration,
are placed on the agar surface
 The Petri dishes are incubated under suitable conditions
 Generally, antimicrobial agent diffuses into the agar a
inhibits germination and growth of the test microorganism
and the diameter s of inhibition growth zones are
measured

37. Contd.
 Inhibition zone edge is formed at the critical time
where a particular concentration of the antibiotic is
just able to inhibit the organism before it reaches an
over whelming cell mass or critical mass.

38. Agar well diffusion method
 Agar well diffusion method is widely used to evaluate
the antimicrobial activity of plant so microbial extracts
 Similarly to the procedure used in disk diffusion
method, the agar plate surface is inoculated by
spreading a volume of the microbial inoculum over the
entire agar surface.
 Then, a hole with a diameter of 6 to 8 mm Is punched
aseptically with a sterile cork borer or a tip, and a
volume (20–100 mL) of the antimicrobial agent or
extract solution at desired concentration is introduced
into the well.

39.  Then, agar plates are incubated under suitable conditions
depending upon the test microorganism.
 The antimicrobial agent diffuses in the agar medium an d
inhibits the growth of the microbial strain

40. Cross streak method
 Cross streak method is used to rapidly screen
microorganisms for antagonism [36]
 The microbial strain of interest is seeded by a single streak
in the center of the agar plate
 After an incubation period depending upon the microbial
strain, the plate is seeded with the microorganisms tested
by single streak perpendicular to the central streak
 After further incubation, the antimicrobial in- teractions
are analyzed by observing the inhibition zone size.

41. Dilution method
 Broth dilution method
Broth micro- or macro-dilution is one of the
most basic anti- microbial susceptibility testing
methods.
 The procedure involves preparing two-fold
dilutions of the antimicrobial agent (e.g. 1, 2, 4,
8, 16and32 mg/mL) in a liquid growth medium
dispensed in tubes containing a minimum
volume of 2 mL (macrodilution) or with
smaller volumes using 96-well microtitration
plate (microdilution)

42. Dilution method
Broth dilution method
.
Broth micro- or macro-dilution is one of
the most basic anti- microbial
susceptibility testing methods.
 The procedure involves preparing
two-fold dilutions of the antimicrobial
agent (e.g. 1, 2, 4, 8, 16and32 mg/mL)
in a liquid growth medium dispensed
in tubes containing a minimum
volume of 2 mL (macrodilution) or
with smaller volumes using 96-well
microtitration plate (microdilution)

43. Contd.
 Then, each tube or well is inoculated with a microbial
inoculum prepared in the same medium after dilution
of standardized microbial suspension adjusted to 0.5
McFarland scale
 After well-mixing, the inoculated tubes or the 96-well
microtitration plate are incubated (mostly without
agitation) under suitable conditions depending upon
the test microorganism

44. Broth dilution

45. Time-kill test (time-kill curve)
 Time-kill test is the most appropriate method for
determining the bactericidal or fungicidal effect.
 strong tool for obtaining information about the
dynamic interaction between the antimicrobial agent
and the microbial strain.

46. Refrence
1. New Anticancer Agents: In Vitro and In Vivo
Evaluation
DANIEL ZIPS1, HOWARD D. THAMES2 and MICHAEL
BAUMANN1,3,4
2. https://www.slideshare.net/DrAnupThorat/evaluation-
of-anticancer-agents-final
3. https://www.slideshare.net/ashwinisomayaji7/screening-
of-anticancer-drugs
4. Review on in vivo and in vitro methods evaluation of
antioxidant activity
5. Md. Nur Alam *, Nusrat Jahan Bristi, Md. Rafiquzzaman

47. Contd..
6. Methods for in vitro evaluating antimicrobial
activity: Areview $ Mounyr Balouirin, Moulay Sadiki,
Saad Koraichi Ibnsouda