3. Cellular cytotoxicity

This presentation details the definition of cell cytotoxicity and cell viability, the difference between the two term and methods of assessment of cells in culture for presence and absence of cytotoxic chemicals or metabolites.

1. Animal cell science & Technology
3. Cell cytotoxicity
ShailendraSingh Shera, Ph.D

2. Measurement of viability and cytotoxicity
Outline
1.Definition of Viability & Cytotoxicity
2.Methods of assessment
a)Dye Exclusion methods
b)Colorimetric Assay
c)Enzyme release assay
d)Fluorometricassay
e)Luminometricassay
3. Practice Questions

3. Definition: Viability and Cytotoxicity
Cellsincultureshouldberoutinelycheckedfortheirhealthstatustoascertaintheirnormal
physiologicalgrowth.Therearevariousmethodstoascertainthehealthstatusofgrowingcells.
Thesearebasedonvariouscellfunctionssuchasenzymeactivity,cellmembranepermeability,cell
adherence,ATPproduction,co-enzymeproduction,andnucleotideuptakeactivity.Beforegoing
further,let’sstartwithdefinition:
What is viability?
Viability of the cells represents the capability of their existence, survival and developments.
What is cytotoxicity?
Chemicals with a capability to destroy cells i.etoxic/ harmful to cells causing either cells death or
deviation from normal physiological behavior.

4. Methods of Assessment
Cellviabilityandcytotoxicityassaysarebasedoncolorimetric,fluorometricandbioluminescent
detectionchemistries.Thetreebelowshowsvariousmodesofassessingviabilityand
cytotoxicity.
Cell viability and
cytotoxicty
Dye exclusion
method
Colorimetric
assay
Fluorometric
assay
Luminometric
assay

5. Dye exclusion methods
Thistechniquesisbasedonexclusionofdyebyviablecells.
Deadcellshavedamagedplasmamembraneandincreasedpermeabilitytodyes.Therefore,Viablecellsremains
unstainedwhereasdeadcellsarestained.
Commondyes:
Trypanblue:Itselectivelystainsdeadcells.Stainingwithtrypanbluefollowedbymicroscopicexaminationon
hemocytometerisfrequentlyusedmethodtodeterminethecellnumberandpercentviabilityinapopulationof
cells.
Deadcellsarestainedblue
ErythrosineB:ErythrosineB,alsoknownaserythrosineorRedNo.3isavitalstain.
Italsohasfluorescentcapacity.
TheprincipleissimilartoTrypanBlue.Itassesmemraneintegrityandpenetratesplasmamembraneofdead
cells.
Fluoresceindiacetate(FDA):Fluoresceindiacetate(FDA)isanon-fluorescentmolecule,whichishydrolysedto
fluorescentfluoresceininlivecells.
Itisalsoavitalstains.
Thehydrolysisisperformedbyesteraseenzymespresentinviablecellsonly.
viablecellsretainthedyeandfluorescewhereasnonviablecellsdonot.Fluoresceindiacetateisnotcytotoxic.

6. Colorimetric assay
Colorimetricassays:MTTassay,MTSassay,XTTassay,WST-1assay,WST-8assay,LDHassay,SRBassay,
NRUassayandcrystalvioletassay.
Principle:
•Itisthemeasurementofabiochemicalmarkertoevaluatemetabolicactivityofthecells.
•Reagentsusedincolorimetricassaysdevelopacolorinresponsetotheviabilityofcells,allowingthecolorimetric
measurementofcellviabilityviaspectrophotometer
MTTassay
TheMTTassayiscolorimetricassaywhichusesreductionofayellowtetrazoliumsalt(3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazoliumbromide,orMTT.
Itisbasedontheabilityofmitochondrialnicotinamideadeninedinucleotidephosphate(NADPH)-dependent
cellularoxidoreductaseenzymestoreducethetetrazoliumdyeMTTtoitsinsolubleformazan,whichhasapurple
color.
Formazancrystalsarethendissolvedusingasolubilizingsolutionandabsorbanceismeasuredat500-600
nanometersusingaplate-reader.
Thedarkerthesolution,thegreaterthenumberofviable,metabolicallyactivecells.
Duringspectrophotometricdetermination,theabsorbancevaluewillincreasewithtime.%viabilityiscalcultaed
as:

7. XTT Assay
Itisbasedonthereductionofayellowtetrazoliumsalt(sodium3´-[1-(phenylaminocarbonyl)-3,4-
tetrazolium]-bis(4-methoxy6-nitro)benzenesulfonicacidhydrateorXTT)toanorangeformazan
dyebymetabolicallyactivecells
XTTissimplyformeasuringproliferationandisthereforeanexcellentsolutionforquantitatingcells
anddeterminingtheirviability.
TheXTTassayisusedtomeasurecellularmetabolicactivityasanindicatorofcellviability,
proliferationandcytotoxicity.
Itisusedforthemeasurementofcellproliferationinresponsetogrowthfactors,cytokinesand
nutrients
https://www.sigmaaldrich.com/technical-documents/protocols/biology/roche/cell-proliferation-kit-xtt-assay.html

8. Enzyme release assays:
Themembraneintegrityofcellscanalsobeassessedbyestimatingtheenzymesreleased.
Lactatedehydrogenase(LDH)hasbeenthemostwidelyusedenzymeforthispurpose.
Thedamaged/nonviablecellswithcompromisedmembraneintegrityreleaseslactate
dehydrogenase.LDHconvertsLactatetopyruvate,generatingNADHfromNAD.TheNADH
interactswiththeprobetogeneratecolorat450nm.
TheamountofcoloredproductformedisdirectlyproportionaltotheLDHactivityinthesample.
TheLDHactivityisdeterminedasNADHoxidationorINTreductionoveradefinedtimeperiod.
Theresultingredformazanabsorbsmaximallyat492nmandcanbemeasuredquantitativelyat
490nm.

9. Fluorometric assay
Fluorometricassays: alamarBlueassay and CFDA-AM assay.
Fluorometricassaysofcellviabilityandcytotoxicityareeasytoperformwiththeuseofa
fluorescencemicroscope,fluorometer,fluorescencemicroplatereaderorflowcytometer.
Fluorometricassaysarealsoapplicableforadherentorsuspendedcelllinesandeasytouse.
AlamarBlue
AlamarBlueCellViabilityReagentisanindigo-colored,non-toxicreagentthatdetects
metabolicallyactivecellsandisusedforthequantitativeanalysisofcellviabilityandproliferation
Resazurin,theactiveingredientofalamarBluereagentuponuptakebylivingcellsisreducedto
resorufin,acompoundthatisredincolorandhighlyfluorescent.
Absorbance-orfluorescence-basedplatereaderareusedtoassesschangeinviability.
Absorbance(detectedat570and600nm)orfluorescence(usinganexcitationbetween530–
560andanemissionat590nm).
Add alamarblue
solution to complete
media directly
Incubate
1-4 hours
Spectrophotometric/
Fluorometric
observation

10. Luminometric assay
Luminometricassays:ATPassayandreal-timeviabilityassay.
ATPassay
•Whencellsdamagedlethallyandlosemembraneintegrity,theylosetheabilitytosynthetize
ATPandtheATPlevelofcellsdecreasesdramatically.
•TheATPassayisbasedonthereactionofluciferintooxyluciferin.Enzymeluciferasecatalyzes
thisreactioninthepresenceofMg
2+
ionsandATPyieldingaluminescentsignal.
•ThereisalinearrelationshipbetweentheintensityofluminescentsignalandATPconcentration
orcellnumber

11. 1. MTT assay is a type of
a.Colorimetric assay
b.Fluorometricassay
c.Emzymerelease assay
d.None of the above
2. What is the property of vital stain?
a.It should kill cells in culture on addition
b.It should be non toxic to cells in culture
c.Both (a) and (b)
d.None of the above
3. Dye exclusion method assays
a.Membrane integrity of cell
b.Enzyme reduction capability.
c.Integrity of DNA
d.None of the above
4. ATP assay is an example of
a.Luminometricmethod
b.Colorimetric method
c.Enzyme release
d.DNA binding
5. Fluoresceindiacetate(FDA) is hydrolyzed by cellular esterase to …………
a.Resazurin
b.Fluorescein
c.Formazan
d.Dimethylsulphoxide
*Practice and Learn Animal cell Science and Technology: Multiple choice question for learning.
Author: ShailendraSingh Shera. Publisher: Amazon Kindle.
Practice questions: MCQs*

12. 1.https://www.sigmaaldrich.com/technical-documents/protocols/biology/roche/cell-proliferation-kit-
xtt-assay.html
2.https://www.thermofisher.com/order/catalog/product/DAL1025#/DAL1025
3.Aslanturk O.S.“In-vitro-cytotoxicity-and-cell-viability-assays-principles-advantages-and-
disadvantages.” Intechopen. DOI: 10.5772/intechopen.71923
Practice MCQs
1. Practice and Learn Animal cell Science and Technology: Multiple choice question for learning.
Author: Shailendra Singh Shera . Publisher: Amazon Kindle.
References & Further reading