ApplicationNoteCell ImagingAutomated Tissue Culture Cell Fixation and Staining in MicroplatesUsing the EL406 Combination Washer Dispenser to Prepare Samples for Imagingwith the Cytation 3 Cell Imaging Multi-Mode ReaderPaul Held Ph. D., Bridget Bishop, and Peter Banks, Ph. D., Applications Department,BioTek Instruments, Inc., Winooski, VTFluorescence microscopy has traditionally been performed on microscope slides, but thereis a growing trend towards the use of 96- and 384-well microplates as this allows greaternumber of samples to be easily processed and automated. This is certainly true in thefield of High Content Screening. Here we describe the use of the EL406 CombinationWasher Dispenser to automate the fixation and staining processes typically used prior tofluorescence imaging.IntroductionKey Words:ImagingCell StainingImmunofluorescenceTissue CultureDAPIPhalloidinBioTek Instruments, Inc.P.O. Box 998, Highland Park,Winooski, Vermont 05404-0998 USAPhone: 888-451-5171Outside the USA: 802-655-4740Email: [email protected] 2013The hallmark of fluorescent microscopy is theuse of specific mouse monoclonal antibodiesto recognize and bind to cellular structures.The antibody is a marvelous tool of remarkableselectivity. It can be used to identify and quantifyalmost any protein in complex biological matrices.It’s use as a fluorescent marker in microscopy datesback to the pioneering work of Albert Coonsdirectly after World War II, where with colleagues,he labeled antipneumococcus antibodies withanthracene isocyanate and thereby made anantibody that could fluoresce under UV light.Today there are myriad fluorescent probes thatemit across the visible to near IR spectrum andcan be used to make fluorescent antibodies.While occasionally primary antibodies aredirectly labeled, more often a labeled secondaryantibody that recognizes the species and thetype of the primary antibody is used to visualizethe structure. The size of the primary antibodyalso allows the binding of multiple copies of thesecondary antibody, whereas the original targetepitope may not, providing a means to amplify thesignal. Antibodies are large macromolar proteinswhich are not cell membrane permeable, thus forfluorescence microscopy applications the cellsmust be fixed and/or permeabilized, typicallyusing formalin or methanol and a detergent.This allows antibodies to access the variousorganelles and their proteins which are of interest.Alterations in cellular morphology can oftenbe followed through the use of actin-specificstains, such as Alexa Fluor 488-phalloidin.Phalloidin is a bicyclic peptide toxin associatedwith death cap Amanita phalloides mushroomsthat specifically binds to cellular actin polymers,while the covalently attached Alexa Fluor 488 moiety allows for fluorescent visualization.Phalloidin binds to actin at the junction betweensubunits; and because this is not a site at whichmany actin-binding proteins bind, most of theF-actin in cells is available for phalloidin labeling .Because fluorescent phalloidin conjugates are notpermeant to most live cells, like antibodies,they require that cells be either fixed and/orpermeablized. Labeled phalloidins have similaraffinity for both large and small filaments and bindin a stoichiometric ratio of about one phalloidinmolecule per actin subunit. Phalloidins have someadvantages over antibodies for actin labeling. Theirbinding properties do not change appreciablywith actin from different species, including plantsand animals. In addition, their nonspecific stainingis negligible and unlike some antibodies they onlybind filament polymers, resulting in good contrastbetween stained and unstained areas. Becausecellular actin polymers serve as cytoskeletalelements fluorescent stains that bind them canbe used to identify cellular cytoplasmic regions.DAPI (4', 6-diamidino-2-phenylindole) is afluorescent stain that binds strongly to A-T richregions in DNA. As such, this stain is specific forthe nuclear regions of the cell, with minor stainingof the mitochondrial DNA. DAPI has the ability topass through intact cell membranes, so it can beused to stain both live and fixed cells, though itpasses through the membrane less efficiently inlive cells and therefore the effectiveness of thestain is lower. When bound to double-strandedDNA, DAPI has an absorption maximum at awavelength of 358 nm and its emission maximumis at 461 nm. Due to its ability to fluorescently stainDNA it is a very popular marker for cell nuclei.
Application NoteRegardless of the antibody and fluorescent dyecombination used in fluorescence microscopy usingmicroplates, a series of aqueous reagent additions andwash steps to remove unbound materials is required forfixing and specific staining with low background. Herewe describe the use of the EL406 Combination WasherDispenser from BioTek Instruments to automate thereagent additions, as well as the wash steps necessaryfor the preparation of cell samples in 96- and 384-wellplates prior to analysis in the Cytation 3 Cell ImagingMulti-Mode Reader. Figure 1 demonstrates a 3-colorstain image of U-2 OS cells using the EL406 CombinationWasher Dispenser.Cell ImagingInstrumentationLiquid HandlingAll cell washes and reagent additions for cell fixation,permeabilization and fluorescent staining were carriedout using the EL406 Combination Washer Dispenser.The EL406 provides full plate washing along with threereagent dispensers in one, compact instrument. Bothperistaltic pump and syringe pump dispensers were used.For expensive biological reagents, such as primary andsecondary antibodies, the peristaltic pump dispenseris optimum as unused portions of the reagent can beeasily retrieved; for inexpensive chemical reagents suchas fixatives and detergents used to permeabalize cells,syringe pumps can be used. In order to maintain sterility,the EL406 Washer Dispenser was placed in a biosafetycabinet and 5 µL cassettes for the peristaltic pump weresterilized by autoclaving prior to use.Cell ImagingFigure 1. Three color staining of a monolayer of U-2 OScells in a 96-well plate. Nuclei were stained with DAPI;F-actin stained with Alexa Fluor 488-phalloidin; and an antimitophillin antibody labeled with Texas Red. The obviousyellow coloring is produced by the superposition of AlexaFluor 488 and Texas Red dyes. Scale bar indicates 80 µm.Cells were imaged using an Cytation3 Microplate Imager(BioTek Instruments, Winooski, VT) configured withDAPI, GFP and Texas Red filter cubes. The imager usesa combination of LED light sources in conjunction withband pass filters and dichroic mirrors to provideappropriate wavelength light. The DAPI filter cube isconfigured with a 377/50 excitation filter and a 447/60emission filter; the GFP filter cube uses a 469/35 excitationfilter and a 525/39 emission filter; while the Texas Redfilter cube uses a 586/15 excitation and 647/57 emissionfilters.Fixed Cell Imaging ProcessesFixationMaterials and MethodsMaterialsNIH3T3, HEK293 and U-2 OS cells were grown inAdvanced DMEM (cat # 12491) from Life Technologiessupplemented with 10% FBS, 2mM glutamine. Blacksided, clear bottom 96-well (cat# 3603) and 384-well (cat# 3712) were from Corning. TopSeal-A (cat # 6050195)adhesive plate sealers were from PerkinElmer. DAPIdihydrochloride stain (cat# D1306), Alexa Fluor 488phalloidin (cat# A12379), Texas red phalloidin (cat #7471) anti-mitofilin monoclonal antibody (cat # 456400),Texas red goat anti-mouse IgG (cat # T862) and DPBS(cat# 14190), were obtained from Life Technologies.Paraformadehyde (cat# P6148) powder was obtainedfrom Sigma-Aldrich, while Triton X-100 (cat # X198-05) wasfrom Baker.2Cells were washed once with 200 µL of DPBS (NaCl 137mM, KCl 2.7 mM, Na2HPO4 10 mM, KH2PO4 7.4 mM) priorto fixation using the EL406 washer manifold. After which100 µL of 4% paraformaldehyde (PFA) solution was addedusing the EL406 syringe pump dispenser manifold. PFAsolution was made daily by dissolving 4 g of powder into100 mL of DPBS warmed to 60 C. Cells were fixed for 10minutes at room temperature followed by 2 washes of 200µL using DPBS.DAPI StainingFollowing the fixation process described above, cellswere permeablized for 10 minutes using 50 µL of DPBScontaining 0.1% Triton X-100 added with the syringepump dispenser. Cell nuclei were then stained with 50 µLof working solution of DAPI. Working DAPI solution wasprepared from a 14.3 mM stock by dilution to 30 µM inDPBS 0.1% Triton X-100. Cells were stained 10 minutesat room temperature followed by 2 washes with 200 µLof DPBS. After aspiration 100 µL of DPBS was added toall wells and the plates sealed using an optically clearTopSeal-A adhesive plate sealer (Perkin Elmer). Preparedplates were kept at room temperature and protected fromlight prior to imaging.
Application NoteCell ImagingTwo Color Staining: Phalloidin and DAPIFollowing the fixation and permeabilization processesdescribed above, Cellular actin was then stained using50 µL of a working solution of either Alexa Fluor 488or Texas Red phalloidin. Alexa Fluor 488 phalloidinworking solution was prepared fresh by dilution of 6.6µM mM stock methanol solution to 30 nM in DPBS. Thestock solution was stored at -20 C until needed. Cellswere stained for 10 minutes followed by a one-cycle washwith 200 µL of PBS 0.1% DAPI Staining could then beperformed according to the process described above.Three Color Staining: Antibody, Phalloidin and DAPICells were fixed and permeabilized according to theprocesses described above. Then, using the peripumpdispenser, 50 µL of primary mouse monoclonal antibody(1:750) dilution in DPBS, 30 mg/mL BSA, 0.1% TritonX-100) specific for mitochondrial protein mitofilinwas added and the plate incubated at room temperaturefor 60 minutes. The plate was washed one time withDPBS 0.1% Triton X-100 and the secondary Texas Redlabeled fluorescent anti- mouse IgG antibody (1:500dilution in PBS, 30 mg/mL BSA, 0.1% Triton X-100)was added and allowed to incubate for 60 minutes atroom temperature followed by a wash with DPBS 0.1%Triton X-100. Following antibody staining, phalloidin,then DAPI staining were performed according to theprocesses described above and evident in the work flowcontained in Figure 2.EL406 Washer umpCell Fixation1 X 200 µLwash100 µLDispenseSyringePermeablization2 X 200 µLwash100 µLDispenseSyringe1 Antibody1 X 200 µLwash50 µLDispensePeripump2 Antibody1 X 200 µLwash50 µLDispensePeripumpPhalloidinstaining2 X 200 µLwash50 µLDispensePeripumpDAPI Staining1 X 200 µLwash50 µLDispensePeripumpFinal Wash1 X 200 µLwash100 µLDispenseWasherTable 1. EL406 Combination Washer Dispenser pump usagefor staining processes.Process96-well384-wellNumber of Cycles1-21-2Soak/ShakeNoNoSoak DurationDispense:Volume2001001CW1CWDispense Height121121Horizontal X Position-150Flow RateHorizontal Y Position00200100TypeHeightTop45Top45Horizontal X Position-500Horizontal Y Position00Delay Vacuum OnAspiration:Rate6CW6CWAspiration Delay00Final Aspiration:YesYesHeight4040Horizontal X Position-500Horizontal Y PositionFigure 2. Automated Workflow for Cell Seeding, Fixation,Permeabilization and Three Color Staining Process. An EL406Combination Washer Dispenser controlled by Liquid HandlingControl (LHC) software was used to carry out the process stepsfor cell fixation, permeabilization and staining with three colors:DAPI nuclear stain, Alexa-Fluor 488 phalloidin actin stain andTexas red labeled secondary antibody.300Rate6CW6CWDelay00Table 2. EL406 Combination Washer Dispenser parameters.
Application NoteCell ImagingResultsFixation and DAPI Staining of CellsCellular expression of transiently transfected genesis a very common experimental procedure. Many ofthese expression experiments use easily traceablegenetic elements (e.g. GFP) either as a direct reporterfor assessment of gene expression or as an internalcontrol for transfection efficiency. Direct single colorimage analysis can be used to identify numbers ofpositively expressing cells (Figure 3). Alternatively,two color blue, green analyses can be used where thenuclei specific blue-DAPI stain can be used to identifyall cells, while the green GFP specific color is usedto identify the positively expressing cells. In eithercase cell fixation can be used to prevent alterationsthat may occur during the image capture process.Figure 4. NIH3T3 cells transfected with GFP. Cellsin 96-well plates were fixed with PFA and stained withDAPI. Image represents an overlay composite of greenand blue fluorescence. Scale bar is 80 µm in length.Two Color Staining of CellsNon-fluorescent cells can also be stained with twodifferent fluorescent stains after fixation. Usually, thisrequires permeabilization of the cell, as is the case herewith the use of phalloidin. PFA fixed cells can be stainedwith both Texas red-phalloidin and DAPI in succession.Peripump cassettes were swapped between reagentadditions such that a fresh cassette was used for eachreagent addition. In Figure 5, all cells have been stainedwith both an actin specific stain as well as a DNA specificstain.Figure 3. GFP expressing NIH 3T3 cells fixed with4% Para-formaldehyde and stained with DAPI. Imagerepresents an overlay composite of brightfield, and bothblue and green fluorescence. Scale bar indicates the size of80 µm.As demonstrated in Figure 3, GFP expressing cellscan be fixed with 4% PFA using the EL406 and GFPfluorescence detected. In this image a manual countof the cells indicates approximately 25% of the cellsare positive for GFP expression. It is important tonote that fluorescent proteins require hydrationso fixation using cold methanol often leads a lossof fluorescence. Image analysis that automaticallycalculates transfection efficiency often requires thatall cells be identified in some fashion. Nuclear stains,such as DAPI, work well to identify total cell number,while analysis of GFP signals identifies positive cells.The ratio of the two values is the transfection efficiency.Figure 4 demonstrates that the EL406 can fix cells andthen add DAPI stain. HeLa cells were first fixed with4% PFA. During the 10-min incubation, the peripumpwas used and DAPI stain added. Manual cell countingindicates that 43% of the total cells are positive for GFPexpression.4Figure 5. Fixed U-2 OS cells in 96-well microplatesstained for F-actin and DNA. F-actin was stained withTexas Red phalloidin, nuclear DNA was identified withDAPI. Image represents the overlay of separately capturedblue and green fluorescent signals. Scale bar represents 80µM.
Application NoteCell ImagingGFP expressing cells can be stained with fluorescentdyes that do not overlap the fluorescent protein’sspectrum. Cells treated with DAPI and Texas redphalloidin will result in a three color fluorescent imagedepicted in Figure 6. Red staining identifies F-actinfilaments and cellular attachment points, while the greensignal denotes GFP located primarily in the cytoplasmand the blue DAPI signal identifies the nucleus. Notethat the GFP signal will often fade much quicker than thesmall molecule fluorescent compounds such as DAPI,Texas red or Alexa Fluor 488 often used. In additionfluorescent proteins require hydration in order to befluorescent, which rules out the use of methanol as afixative agent.Figure 7. U-2 OS cells stained for mitochondria, F-actinand nucleus. Cells in 96-well plates were fixed and stainedfor mitochondria (Texas Red), F-actin (Alexa Fluor 488), andDNA (DAPI). Scale bar indicates 80 µm.Higher density plates are being used more often inconjunction with high content screening. The EL406 iscapable of processing 96- and 384-well plates withoutchanging manifolds. As demonstrated in Figure 8, HeLacells expressing GFP cultured in 384-well microplates canbe fixed and washed using the EL406. Cell monolayerswere intact and cells uniformly fluorescent.Figure 6. NIH 3T3 cells expressing GFP. F-actin wasstained with Texas Red-phalloidin and nuclear DNA wasidentified with DAPI. Image represents the overlay ofseparately captured red, blue, and green fluorescentsignals. Scale bar represents 80 µm.Three Color Staining of CellsThe sequential addition of multiple stains, each specificfor a separate cellular target and with a distinguishablesignal is a commonly used fluorescence microscopytechnique. One is only limited by the ability to identifyspecific targets and the capability of the imaginginstrument being used. Figures 7 demonstrates theability of the EL406 to fix and permeabalize U-2OS cells and then add a primary antibody specific tomitochondrial proteins, along with a fluorescent labeledsecondary detection antibody that recognizes theprimary antibody and two other stains. In this three-colorimage mitochondria are identified by the red signal,cytoplasmic F-actin identified by green and nuclei byblue signals.5Figure 8. HeLa-GFP cells in 384-well microplates fixedwith 4% PFA. Scale bar indicates 1000 µm.
Application NoteLikewise, cells can be stained with DAPI. As shown inFigure 9, NIH3T3 cells show uniform staining for nuclei.Figure 9. NIH3T3 cells expressing GFP in 384-wellmicroplates. Cells were fixed with 4% PFA, and stainedwith DAPI. Scale bar indicates 400 µm.DiscussionThese image data indicate that the EL406 CombinationWasher Dispenser can be used to automate the cellfixation, permeabilization and staining processes usedin many fluorescence microscopy experiments usingmicroplates, including High Content Screening (HCS).Automation of the sample preparation steps of theseexperiments allows for a significant increase in thenumbers of samples that can be analyzed. In addition,automation provides a level of consistency not seen withmanual processing.Most staining procedures require that the cells bepermeablized with some sort of detergent, such asTriton X-100. Large molecules such as antibodies andphalloidins will not cross intact membranes. WhileDAPI is reported to cross membranes and work on nonpermeablized cells, it does so much less efficiently thancells that have been permeablized. We routinely useTriton X-100 with DAPI staining to insure adequate stainpenetration. Because of their high specificity, brightness,physical robustness and large amount of target,phalloidin and DAPI staining can often be combined intoa single step, saving time and subjecting the cells to lesswashing.The numerous process steps required to fluorescentlystain cells with three colors can be modified extensivelydepending on the needs of the researcher. Manyexperimental questions do not require three colorstaining. For example, measuring transfection efficiencyof GFP expressing cells may only require a DAPIfluorescent stain, after fixation with 4% PFA.6Cell ImagingPositive cells will produce green fluorescence, while allcell nuclei will be stained blue with DAPI. Alternatively,cell morphology can be identified with fluorescentphalloidin stains specific to actin and DAPI stain willidentify nuclei. Nuclear translocation assays need onlyhave a stain identifying the protein in question and DAPIto identify the nucleus of the cell. The individual processsteps can be used or not used as experimentally needed.At the end of the required process, a final wash to removeunbound stain or antibodies followed by the addition ofPBS to keep the samples hydrated is all that is necessary.The EL406 Combination Washer Dispenser has a numberof features that make it amenable for the processsteps of fixing and staining tissue culture cells prior tofluorescence microscopy. Typically cells are washedwith PBS or similar isotonic buffer between reagentadditions to remove unwanted media or to eliminateunbound antibodies or dyes. The washer manifold hasa full 96 head that is capable of rapidly washing either96- or 384-well microplates without changing washermanifolds.In addition, the combination of syringepump and peripump dispensers allows for the additionof different reagents. Use of the peripump dispenser(Table 1) in conjunction with a 1 µL cassette allows theuser to minimize the amount of overage required for anadequate dispense. Unused precious reagent can alsobe recovered by reversing the peripump and purgingthe lines back into the reagent container. Additionally,the multiple reagent paths of the peripump can be usedto add different antibodies or stains to different wellson the same microplate; thus allowing for side by sidecomparisons without the need for subsequent reagentadditions. The syringe pumps allow for different reagents(e.g. PFA fixative and permeabilization buffer) to beadded using separate fluid paths without any manualintervention.Automation of the staining process steps requires theability to gently add and remove fluids from microplatewells without physically damaging the cells in the wells.As described previously physical trauma to cells canresult from using too vigorous of a dispense rate . Thisis particularly true of the wash buffer being dispensedfrom 96-tubes simultaneously. Leaving a fluid residualserves to minimize the impact of incoming washer buffer.The residual used needs to be balanced with the dilutionto any subsequent reagent added. Towards that endwe used physical offsets to move the dispense tubes forthe washer manifold to one edge of the wells (Table 2).Likewise having the aspiration tubes in close contact withthe cell layer for an extended period of time is harmfulto the cell monolayer. A small yet strong vortex occursnear the aspiration tube that can damage fixed or livecells. The use of rapid aspiration dive rates minimizesthe exposure of cells to this deleterious force (Table 2).
Application NoteCell ImagingHCS has started to play a significant role in the drugdiscovery process, beyond secondary screening. Bymarrying the wealth of information accessible throughmicroscopy with large numbers of experimentalconditions in a single experiment, unique pieces ofinformation can be gathered that might have beenmissed with either a traditional cell based screen or amanual microscopic study. Microscopic image basedanalysis often requires significant amount of manualsample processing, which can be tedious and subjectto process errors that can result in artifacts. The use ofautomation for these steps, besides being faster alsoeliminates any technician bias of the data.References1. Steinmetz, M. O., Stoffler, D., Müller, S. A., Jahn, W.,Wolpensinger, B., Goldie, K. N., Engel, A., Faulstich, H.& Aebi, U. (1998) Evaluating atomic models of F-actinwith an undecagold-tagged phalloidin derivative. J.Mol.Biol. 276, 1-62. Held. P. (2005) BioTek Application Note ely-adherenttissue.htmlAlexa Fluor is a registered trademark of Molecular Probes Inc.7AN041013 15, Rev. 08/13/13