BasicProcedure
WashingCellsinSeahorseXFpCellCultureMiniplates
BeforeperforminganXFassay,growthmediummustbereplacedwithasuitableassaymedium(generallythis
meansmediumwithoutbicarbonatebufferorserumandwithlowphenolredcontent).Thisproceduredescribes
replacingthegrowthmediumwithassaymediumforadherentcellsgrowninSeahorseXFpCellCultureMiniplates
priortobeingassayedusingaSeahorseXFpAnalyzer.
MaterialsRequired:
• Preparedassaymedium.SeetheBasicProcedureforAssayMediaPreparationfordetailsonchoosingand
preparingtheassaymedium.
• Multi-channelpipette,200μLcapacity,withmatchingtips
• Tissueculturemicroscope
• Non-CO2incubator
Procedure:
1. Warmtheassaymediumto37°C.
2. RetrievetheSeahorseXFpCellCultureMiniplate(s)fromthetissuecultureincubator.Youmaywishtokeepthe
miniplate(s)intheSeahorseXFpCarrierTrayforeaseofhandling.
3. Lookatthecellsunderthemicroscopeto:
a. Confirmcellhealth,morphology,andpurity(nocontamination).
b. Ensurethatthecellsareadheredandappearasaconsistentmonolayer.
c. Makesurethatthebackgroundwells(AandH)containnocells.
4. Washthecellswithassaymedium:
a. Removeallbut20μLoftheculturemediumfromeachwell.Thesmall
amountofmediumislefttokeepthecellsfromdryingout.
b. Gentlyaddapproximately200μLofassaymedium,thenremovethe
Incubatingthecell
sameamount.
plateswithoutCO2
c. Repeatstep4b,removingallbut20μL(asinstep4a).
allowsoutgassing
d. Addassaymediumtoatotalvolumeof180μL–orthevolume
fromtheplateandis
recommendedbytheparticularassayprotocolyouareusing.
requiredforaccurate
5. Observetheassaywellsunderthemicroscopetoensurethatcellswerenot
ECARmeasurements.
washedaway.
6. Placetheplateina37°CincubatorwithoutCO2foronehourpriortotheassay.