Forlifescienceresearchonly.
Notforuseindiagnosticprocedures.
DAPI(4’,6-Diamidine-2’-phenylindole
dihydrochloride)
Powder
Cat.No.10236276001
y
Version17
Contentversion:January2007
10mg
Storethiskitat+15to+25°C
1.
Productoverview
Formulation
Powder(crystallized);
Properties
Formula
C
16
H
15
N
5
×2HCl
Molecularweight
M
r
350.3
Solubilityinwater
25mg/ml
Absorbancemaximumin
aqueoussolution
ϭ
340nm
Emissionmaximumin
aqueoussolution
ϭ
488nm
Typicalanalysis
Ͼ
9
0%(fromN.)
Application
Detectionofmycoplasmalinfectionsofcellcultures.
Assayprinciple
ThefluorescentdyeDAPIbindsselectivelytoDNAand
formsstronglyfluorescentDNA-DAPIcomplexeswith
highspecificity.
OnaddingDAPItotissueculturecellsitisrapidly
takenupintocellularDNAyieldinghighlyfluorescent
nucleiandnodetectablecytoplasmicfluorescence.If
thecellsarecontaminatedwithmycoplasmas,charac-
teristicdiscretefluorescentfociarereadilydetected
overthecytoplasmandsometimesinintercellular
spaces.
Reconstitution
In2–10mldoubledist.water;1–5mg/mlfinalconcen-
tration.
Note
:Preparealiquotsandstoreat
Ϫ
15to
Ϫ
25°C.
Storagestability
Stableat+15to+25°C,protectedfromlight,untilthe
expirationdateprintedonthelabel.
Solution
Storage/stability
Stocksolution(1–5mg/ml)
Ϫ
15to
Ϫ
25°Cfor
12months
Workingsolution(1
g/ml)
+2to+8°Cforabout6
months
Background
information
Figuresontheincidenceofmycoplasmalinfectionsof
cellculturesrangefrom1–92%(1-4).Theoriginsof
mycoplasmalinfectionofcellculturesarebovine
serum
(A.laidlawii
,
M.arginini
,
M.hyorhinis
),laboratory
personnel(
M.orale
)andmycoplasma-infectedcul-
tures.
Mycoplasmasproducevariouseffectsontheinfected
cellculture(2–4).Mycoplasmalinfectioncannotbe
detectedbynakedeyeotherthanbysignsofdetoria-
tionintheculture.Itisimportanttoappreciatethat
mycoplasmasdonotalwaysrevealtheirpresencewith
macroscopicalterationsofthecellsormedia.Many
mycoplasmacontaminants,particularlyincontinuous
celllinesgrowslowlyanddonotdestroyhostcells.
Thereforethereisanabsoluterequirementforroutine,
periodicassaysforpossiblecovertcontaminationofall
cellcultures,particularlycontinuousorestablishedcell
lines.
Detection
techniques
Avarietyoftechniqueshavebeendevelopedforthe
detectionofcellculturemycoplasmas,e.g.
•
DNAstaining,
•
mycoplasma-mediatedcytotoxicity,
•
biochemicaldetectionmethods,
•
electronmicroscopy,
•
ELISA(MycoplasmaDetectionKit*)orbyPCR
(MycoplasmaPCRELISA*)(2,3,5).
DNAstainingemployingfluorescentdyesthatbind
specificallytoDNAisthemostpopularmethod.This
methodisquickandsimpletoperform.Twodyes,4’,6-
diamidine-2’-phenylindole(DAPI)andbisbenzimide
(H33258)havebeenwidelyused(6–8).Therationale
behindthisassayisthatmycoplasma-freecultures
exhibitonlynuclearfluorescence.Mycoplasma-
infectedculturesalsodisplayextranuclearfluores-
cence.MitochondrialDNAisnotapparentinprepara-
tionsstainedeitherwithDAPIorH33258.
2.
Proceduresandrequiredmaterials
2.1Beforeyoubegin
General
considerations
Beforetheassaycellculturesshouldbepassedinanti-
biotic-freemediaforaminimumoftwopassages.
Theculturesshouldbeassayed3–4daysafterpas-
sage.Thecellsupernatantwillcontain10
7
to10
8
CFU/
ml,additionalorganismareadsorbedontohostcells.
Preparationof
stocksolution
Dissolveindoubledist.watertoafinalconcentration
of1–5mg/ml.
Note
:Donotuseanybuffers.
Preparationof
workingsolution
Dilutethestocksolutionwithmethanoltoafinalcon-
centrationof1
g/ml.Theworkingsolutionisstableat
+2to+8°C,forabout6months.
2.2
Stainingofmonolayercultures
Procedure
Pleasefindtheprotocolforthestainingofmonolayer
culturesinthefollowingtable.
Step
Action
1
Allowculturestoreach50–70%confluence.
Note
:Allowingculturestoreachconfluencewillimpairsubsequent
visualizationofmycoplasmas.Culturesmaybegrownoncoverslipsin
petridishes.
2
Pouroffthemediumfromthecells.
3
WashoncewithDAPI-methanol(workingsolution,1
g/ml).
4
CoverthecellswithDAPI-methanolandincubatefor15minat37°C.
5
Pouroffthestainingsolution.
6
Washoncewithmethanol.
7
Placetheinvertedcoversliponamicroscopeslide,usingglycerolor
PBSasmountingmedium,avoidwater.
Examineunderafluorescencemicroscopewith340/380nmexcitation
filterandLP430nmbarrierfilter(e.g.Leitzfiltercombination:BP340–
380,RKB400,LP430;Zeissfiltercombination:BP365/11,FT395,LP
397orBP340–380,RKP400,LP430).
Atotalof500×(40×12.5)magnificationisgenerallysufficientin
detectingbrightlyfluorescentmycoplasmas.
Butbestresultsareobtainedusinga100×oilimmersionobjective.
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