Journal of Microbiological Methods 94 (2013) 111–116
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Journal of Microbiological Methods
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Improvement of 96-well microplate assay for estimation of cell growthand inhibition of Leishmania with Alamar Blue
María Jesús Corral, Elena González, Montserrat Cuquerella, José María Alunda ⁎Department of Animal Health (ICPVet Group), Faculty of Veterinary Medicine, Universidad Complutense de Madrid, 28040 Madrid, Spain
The value of resazurin-based Alamar Blue redox indicator to determinemultiplication of Leishmania promastigotesin 96-well microtiter plates was examined. In addition, assaywas validatedwith amphotericin B (AmB) and allicin.The method was tested on L.donovani and L.infantum promastigotes under different culture conditions (variableair-phase, presence of phenol red, initial cell density, incubation time, use of Hepes buffer). Results showed thatthe gas-phase of promastigote cultures was critical. The method yielded consistent results with initial plating celldensities of 2.5 × 105 promastigotes/well, up to 72 h incubation and 5% CO2 atmosphere or reduced air availability(sealed plastic bags, film-sealed microplates). Detection of low numbers of promastigotes and earlier results couldbe obtained using fluorimetry instead of spectrophotometry. The addition of 20 mM Hepes improved the results.Fluorescence intensity correlated to promastigotes number in both Leishmania spp. Inhibitory concentration(IC50) values for AmB and allicin using cell counting and fluorimetry were comparable. Under these conditionsthis one-step, low-cost redox indicator can be used in drug sensitivity assays and studies of differential proliferationrates of Leishmania isolates or strains in a 96-well format.
Visceral leishmaniasis is a parasitic disease caused by Leishmaniadonovani and L. infantum (= L.chagasi) (Kinetoplastida). The infectionaffects both humans and dogs in large areas of the world (i.e. India,Mediterranean Basin, and South America) and it is fatal unless treated.Current first-line chemotherapy of leishmaniasis relies on a rather lim-ited arsenal of drugs, most of which have serious side-effects includingnephro- and hepatotoxicity and teratogenicity. Therefore, the identifi-cation of new molecules or formulations is an urgent need and hasbeen recognised byWHOas one of the research areaswhere a sustainedeffort has to be made (Alvar et al., 2006).
It is assumed that in vivomodels have superior predictive value thanin vitro models, and that screening using intracellular amastigotes aremore convenient than axenic amastigotes and promastigotes (Serenoet al., 2007; Vermeersch et al., 2009; De Muylder et al., 2011; Guptaand Shakya, 2011). In spite of the limitations the promastigote stage iscurrently used and screeningwith this parasitic stage has been exploitedas a first-step to identify “hit” and “lead” anti-leishmanial compounds inundirected massive screening [High Throughput Screening (HTS)] ofchemical libraries (Sharlow et al., 2009; Siqueira-Neto et al., 2010;Walkeret al., 2011).
culty of Veterinary Medicine,Spain. Tel.: +34 91 3943701;
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There are several in vitro systems available to determinepromastigotes proliferation of Leishmania spp (i.e. reporter geneassays, enzymatic determinations, H3 -thymidine incorporation, colori-metric methods). Among colorimetric methods, resazurin-based AlamarBlue entails several advantages. First, it is simple to use as it requires onlya one-step procedure. Other benefits reported are its low cost, environ-mentally friendly composition and transferability to field sites if neces-sary (Räz et al., 1997). Unlike other assays, this redox indicator isrelatively non-toxic to cells and can be usedwith long incubation periods(up to 72 h) (Fumarola et al., 2004). This indicator has been extensivelyused in the related genus Trypanosoma (Räz et al., 1997; Rolón et al.,2006; Sykes and Avery, 2009) and in some Leishmania spp (Mikus andSteverding, 2000; de Oliveira-Silva et al., 2008; Shimony and Jaffe,2008; Kulshrestha et al., 2013). Recently, this method has been adaptedusing HTS with Leishmania and two different platforms and a 384-wellformat (Sharlow et al., 2009; Siqueira-Neto et al., 2010).
Our laboratory has been engaged on the study of the anti-leishmanialantiproliferative effect of differentmolecules.While Alamar Blue could beeasily employed to determine the cytotoxicity for the murine cell lineJ774 (Wert et al., 2011), results obtained with Leishmania promastigoteswere inconsistent, since resazurin reduction did not correlate with cellcounts. Given the lack of experimental details given in the available liter-ature dealing with Leishmania, our aim was to examine the value ofAlamar Blue to determine the multiplication and growth inhibition ofL. donovani and L. infantum promastigotes under different culture condi-tions (variable air-phase, cell density and incubation time). Resultsshowed that optimal conditions of Alamar Blue assaywith promastigotesin 96-well microtiter plates included a 5% CO2 atmosphere, the presence
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on 20 mM Hepes in the culture medium and an initial concentrationof promastigotes of ca. 2.5 × 105/mL. With these conditions, reducedresazurin (resorufin) measured by fluorimetry provided an accurateestimation of promastigotes multiplication and could be used for drugscreening and IC50 estimation.
2. Material and methods
2.1. Parasites
An autochthonous isolate of L. infantum (UCM 9), obtained fromaffected dogs in the area of Madrid (Spain) by the Clinical Serviceof the Department of Animal Health, Faculty of Veterinary Medicine(Universidad Complutense), and Khartoum 1246 isolate fromL. donovani, provided by Dr. Toraño (Department of Immunology,Instituto de Salud Carlos III, Madrid) were routinely maintained aspromastigotes in RPMI 1640 medium (Lonza Group, Basel, Switzerland)at 26 °C supplemented with heat inactivated (30 min, 56 °C) foetalbovine serum (FBS) (Sera Laboratories International, Horsted Keynes,UK) and 100 U/mL penicillin + 100 μg/mL streptomycin (BioWhittaker,Verviers, Belgium) in 25 mL culture flasks.
2.2. Chemicals
Alamar Blue was purchased from AbD Serotec (Oxford, UK). Allicin(2-Propene-1-sulfinothioic acid S-2-propenyl ester) was obtained asliquid Allisure® from Allicin International Ltd (Rye, East Sussex, UK)at a concentration of 5000 ppm and kept at a temperature of −80 °Cuntil used. Amphotericin B (AmB) was obtained as fungizone (Sigma,St. Louis, USA).
2.3. Promastigote assays
Depending on the experiment promastigotes were cultured inflat-bottomed 96-well cell culture microtiter plates with lid (Costar,Corning, NY, USA), in microtiter plates wrapped with Parafilm®, orin plates sealed with Thermal adhesive film for PCR plates (Simport,Beloeil, Canada). For comparative purposes promastigote cultures werealso done in 1.5 mL eppendorf ® tubes. Cultures were carried out at26 °C in aerated culture chamber or incubated in a 95% air/5% CO2
humidified atmosphere. Culture media (RPMI 1640) with and withoutadditional 20 mMHepes were employed depending on the experiment.
2.4. Alamar Blue assay
Concentration of resorufin, the product of reduction of resazurin, inthe Leishmania cultures was determined following the manufacturer'srecommendations by reading the absorbance (A) at 570 and 600 nm,and fluorescence (550 nm excitation wavelength, 590 nm emissionwavelength) in a FLUOstar Omega (BMG Labtech, Ortenberg, Germany)fluorimeter. Fluorescence intensity was expressed as arbitrary units(A.U.). Briefly, mid-log phase promastigotes were added to the wellsof microtiter plates or eppendorf tubes up to a volume of 200 μL/well.After 24 h, 20 μL Alamar Blue (10% v/v) was added and the cultureswere kept for 24, 48 or 72 additional hours. Absorbance and fluores-cence intensity were determined every 24 h. Promastigote countswere carried out in Neubauer improved chambers and cell viabilitywas assessed by trypan blue exclusion staining. Untreated cultures,wells without cells and the maximal concentration of the drugs, andwells with culture medium and Alamar Blue (10% v/v) were includedas controls. All experiments were performed at least in triplicate.
2.5. Statistical analysis
Results were expressed as means ± standard deviation. Data werecompared by analysis of variance (one- and two-ways ANOVA) and
GLManalysis using GraphPad Prism5. Differenceswere considered signif-icant when p b 0.05. Figures were also prepared with GraphPad Prism5.
3. Results
3.1. Determination of optimal cell density
Different concentrations (104, 2.5 x104, 5 × 104, 7.5 × 104, 105,2.5 × 105, 5 × 105, 7.5 × 105, 106) of mid-log phase promastigotes ofL.donovani and L.infantum were added in a final volume of 200 μL/wellin 96-microtiter plates. Cultures were carried out at 26 °C in plateswith lid under a 5% CO2 atmosphere or in film-sealed plates. After24 h incubation, Alamar Blue was added and the plates were kept for24, 48 or 72 h. For each time determination a plate was used. Resazurinwas effectively reduced to resorufin in the medium, evidenced by thehigher levels of absorbance and, particularly, fluorescence related bothto the initial promastigotes density and the time of culture. Absorbancedeterminations, especially with low initial promastigote concentration(b105 promastigotes/well) were more variable (not shown). This vari-ability was not observed when cultures were kept in the CO2 atmo-sphere. Results were more consistent when resorufin concentrationwas determined by fluorimetry. Fluorescence was significantly higher(ca. 2 times) when cultures were exposed to 5% CO2 (Fig. 1A) as com-pared to those performed in film-sealed plates (Fig. 1B). Highest levelsof fluorescence were obtained with initial inoculums of 2.5 to 5 × 105
promastigotes/well from both Leishmania species, after 72 h and expo-sition to CO2.
3.2. Correlation between Alamar Blue reduction andpromastigotes multiplication
Preliminary results obtained in our laboratory allowed the use ofthe redox indicator to determine the proliferation of Leishmaniapromastigotes in culture tubes. However, as shown above, for a given ini-tial number of promastigotes and time of incubation, significant differ-ences (p b 0.05) were found in the concentration of resorufin estimatedby absorbance and, particularly, fluorimetry depending on the expositionto CO2 or to a limited air phase in the sealedmicrotiter plates. To rule outthe possibility of resazurin reduction being an inaccurate estimation ofLeishmaniamultiplication, promastigotes (2.5 × 105/well) were culturedin 96-well plates under a CO2 atmosphere or under air phase. For compar-ative purposes parallel cultureswere done in eppendorf tubes. In all casescell multiplicationwas estimated by fluorimetry and cell counting of via-ble Leishmania in Neubauer chamber. A set of cultures was employed foreach time determination (24, 48, 72 and 96 h).
Fig. 2 shows that no significant differences (p > 0.05) were foundbetween cultures in the experiment irrespective of the exposition to5% CO2 or to an unlimited air phase. After 96 h, all cultures reachedvalues ca. 2.7 × 106 promastigotes/mL in both Leishmania spp (Fig. 2A).However, resazurin concentrationdeterminedbyfluorimetrywas strong-ly dependent on the culture conditions (Fig. 2B). Highest levels of fluores-cence were seen in cultures exposed to CO2 and the lowest values werepresent using standard culture conditions with air atmosphere. Culturesin eppendorf tubes, with a limited amount of air, displayed intermediatevalues. These results were consistent with the absence of colour changein the standard microtiter plates in spite of the active multiplication.These results suggested the importance of the air phase of the culturesin the usefulness of Alamar Blue method to determine Leishmania prolif-eration and also the need of standardization of the assay.
3.3. Effect of atmosphere and buffer on resazurin reduction bypromastigotes of Leishmania
Promastigotes of L.infantum and L.donovaniwere cultured in 96-wellstandard microtiter plates exposed to 5% CO2, in standard microplates,in microplates wrapped with Parafilm, in microplates in sealed plastic
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Fig. 1. Effect of incubation time with Alamar Blue on the fluorescence curves with promastigotes of L.donovani. Increasing initial plating concentrations of promastigotes were incubatedwith Alamar Blue (10% v/v) and the fluorescence intensity determined (arbitrary units, A.U.) (excitationwavelength 560 nm, emissionwavelength 590 nm). A: In the presence of 5% CO2.B: Film-sealed microplates. ●: 24 h; ■: 48 h and ▲: 72 h. Results are means ± standard deviations of 3 determinations.
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bags or film-sealed microplates for PCR. Moreover parallel cultureswere done in eppendorf tubes to monitor cell viability (trypan Blue ex-clusion dye) and multiplication (counting in Neubauer chamber). Cul-tures with standard RPMI medium and medium with 20 mM Hepeswere employed. Absorbance and fluorescence were determined 24, 48and 72 h after Alamar Blue addition.
As expected time-dependent fluorescence increases were observed inall culture conditions and both Leishmania species earlier than absorbancevariations (not shown) in the determinations carried out. Fig. 3 shows theresults obtained with L. infantum and similar values were obtained forL.donovani. It was found that the presence of 5% CO2 or limited air avail-ability was critical to get the highest fluorescence values. Microplateswith lid exposed to air had the lowest levels, comparable to those foundin Parafilm wrapped plates. By its part, 96-well plates cultured in a CO2
incubator displayed the highest resorufin concentrations in all timedeter-minations carried out. In cultures done in eppendorf tubes, film-sealedmicroplates and plastic-sealed microplates intermediate values of ab-sorbance and intensity of fluorescence were obtained. Moreover, thepresence of additional 20 mMHepes improved the resazurin reductionin all culture conditions, particularly when film-sealedmicrotiter platesand eppendorf tubes were employed (p b 0.01 – p b 0.001).
3.4. Value of resazurin reduction measured by fluorimetry for drugscreening in Leishmania
Value of resazurin transformation by fluorimetry for drug screen-ing in 96-well microtiter plates was tested with two compounds with
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Fig. 2. A: Proliferation of Leishmania promastigotes under different culture conditions [WPromastigotes were seeded (2.5 × 105 promastigotes/well) on day 0. Alamar Blue was added 2at different times and culture conditions ○: 5% CO2 atmopshere;●:aerated microplates; ▲:ep
antileishmanial activity, namely allicin and AmB. In all cases 2.5 × 105
promastigotes/well for both species were used. In the case of allicin,10, 30, 60 and 120 μM concentrations were used and microplates inCO2 or aerated incubator, and with and without additional 20 mMHepes in themedium, were used. Parallel counts were done in Neubauerhaemocytometer with cultures in eppendorf tubes exposed to the sameallicin concentrations. Cultures were treated for 24 h with allicin andabsorbance and fluorescence were monitored at 24, 48 and 72 h afterexposure to the drug. A range of AmB (0, 0.001, 0.002, 0.003, 0.004,0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07,0.08, 0.09, 0.1, 0.5, 1 and 10 μM) was tested with the optimal assay con-ditions found (5% CO2, 20 mM Hepes). Fluorescence and absorbancewere measured 72 h after exposure to the antibiotic.
Fig. 4A shows the relationship between the allicin concentrationand the growth inhibition of L.donovani promastigotes determined bycell counting and fluorescence of the cultures. Addition of 20 mMHepes improved the detection of fluorescence in aerated microplateswhereas no correlation between growth inhibition and resorufin levelsin the cultureswas observedwithoutHepes in this case. Our results clear-ly showed that Alamar Blue reduction closely correlated to microscopiccell counts and viability, in the allicin concentration range examined,provided that a 5% CO2 atmosphere and 20 mM Hepes were present(Fig. 4B). Comparable results were obtained for L.infantum. Inhibition ofmultiplication of Leishmania promastigotes in the presence of AmBconfirmed the value of the resorufin level with fluorimetry using theassay conditions described in the screening and IC50 determination ofthis antileishmanial drug. Actually, for the particular L.infantum isolate
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pendorf tubes. Results are means ± standard deviations of 3 determinations.
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Fig. 3. Fluorescence intensity (A.U.) of promastigote cultures of L.donovaniwith addi-tional buffer (20 mM Hepes) at different times of incubation. All cultures except □were carried out in 96-well microplates. ●: 5% CO2 atmosphere; ■:aerated microplates;▲: Parafilmwrapped plates;○: plastic bag-sealedmicroplates;Δ: Film-sealedmicroplates;□: eppendorf tubes. Results are the means ± standard deviations of 3 experiments.
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Fig. 5. Determination of IC50 of amphotericin B for L.donovani promastigotes usingAlamar Blue reagent. Experimental conditions included an initial plating concentrationof 2.5 × 105 promastigotes/well, 5% CO2 atmosphere and 20 mMHepes. Results are themeans ± standard deviations of 3 experiments.
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employed an approximate IC50 of 0.07 μM was obtained by cellcounting>whereas using the best-fit valueswithfluorescence determi-nations the value obtainedwas 0.0618 μM(Fig. 5). Fig. 6 shows a repre-sentative result of the effect of CO2 and 20 mMHepes on the reductionof resazurin.
4. Discussion
Present results, with the optimal conditions described, show thatAlamar Blue, and presumably other resazurin-basedmethods, is an accu-rate assay to determine the multiplication of Leishmania promastigotesand growth inhibition elicited by antileishmanial drugs. Assay conditionsexamined in a checkerboard manner showed that several factors wererelevant to obtain consistent results, namely an initial plating density ofpromastigotes ca. 2.5 × 105, besides the presence of a 5% CO2 atmosphereand additional buffering of the medium with 20 mM Hepes. Our resultson the optimal cell density were comparable to the findings by Mikusand Steverding (2000) with L.major and Shimony and Jaffe (2008) withL.donovani and support the lower reduction rate of Leishmania comparedto T. cruzi epimastigotes (Rolón et al., 2006) and bloodstream trypano-somes (Räz et al., 1997). A linear fluorescence response was observedwith lower plating densities of promastigotes and therefore the methodcould probably be used in 72 h experiments in the range of 5 × 104 to105 promastigotes/well. The plateau observed with higher cell densities
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Fig. 4. A: Growth inhibition of L.donovani promastigotes in the presence of allicin (24 h) estsphere and 20 mM Hepes; □: 5% CO2 atmosphere; ●: aerated microplates and 20 mM Hepinhibition of L.donovani promastigotes, determined by fluorescence and cell countings. ▲: cemicroplates and 20 mM Hepes; ○: aerated microplates. Results are means ± standard dev
and incubation time are probably related to the conversion of resorufinto colourless and non-fluorescent hydroresorufin (O’Brien et al., 2000).
Alamar Bluemethod has been successfully employed tomonitor thegrowth of mammalian cells (Ansar Ahmed et al., 1994; Nakayama et al.,1997; O’Brien et al., 2000; Sykes and Avery, 2009). Most of the assayshave been performed in a 95 % air/5 % CO2 atmosphere, includingthose carried out in African trypanosomes (Räz et al., 1997; Sykes andAvery, 2009), or in plastic sealed bags (Martin et al., 2003). More thanprobably the performance of a redox indicator added to a culture medi-umwould be affected by the gas-phase although this factor apparentlyhas not been considered in the previous contributions on Leishmania(de Oliveira-Silva et al., 2008; Shimony and Jaffe, 2008; Vermeerschet al., 2009; Kulshrestha et al., 2013) and T. cruzi epimastigotes (Rolónet al., 2006) using 96-well microtiter plates or, more recently with theHTS with 384-well format, using L.major (Siqueira-Neto et al., 2010).Our results showed that, using the 96-well plate format incubation ina 5 % CO2 gas phase yielded accurate results of cell growth and inhibi-tion in Leishmania (r2 = 0.9713). The carbon dioxide dissolved in themedium allowed the efficient reduction of resazurin to the fluorescentresorufin. Actually, O’Brien et al. (2000) highlighted the need of CO2 inthe medium when reduction is occurring in order to capture electrons.In the absence of 5% CO2 additional buffering with Hepes could circum-vent some of the limitations provided that a limited volume of gasphase was present (capped eppendorf tubes, film sealed plates, sealedplastic bags). Earlier and more sensitive estimation of resorufin wasobtained by fluorimetry and thus should be preferred to spectropho-tometry. The significantly higher sensitivity of fluorimetry allowed the
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Fig. 6. Representative image of L.infantum and L.donovani promastigote culturesexposed to different concentrations of allicin (0, 10, 30, 60 and 120 μM),with andwithout20 mM Hepes, in the presence of 5% CO2 atmosphere (B) or in air exposed 96-wellmicroplates (A). Marginal wells were not used to avoid edge effect.
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earlier identification of anti-leishmanial activity of allicin. Promastigotemultiplication and inhibition could be easily detected by fluorescenceafter 48 h whereas observation of significant absorbance variationsneeded 72 h as observed by Mikus and Steverding (2000).
Visceral leishmaniasis, both human and animal, caused by L.donovaniand L.infantum (=L.chagasi) continues being a challenge formedical doc-tors and veterinary clinicians in many regions of the world. Availabledrugs have important shortcomings (nephrotoxicity, hepatotoxicity, gas-trointestinal disturbances) and, moreover, in some areas Leishmaniastrains resistant to first line drugs have been described (Croft et al.,2006). Identification of new potentially effective drugs or preparationsrequires robust and reliable screening methods. In vivo tests of efficacymust be reduced at a minimum by both ethical and economic reasonsand therefore in vitro screening of potentially useful compounds isthe first step to identify “hits and leads”. Obviously, the best in vitromodel to test antileishmanial activity of compounds is done on the intra-cellular amastigote within macrophages (Sereno et al., 2007). However,intracellular amastigotes screening is only available to some laboratories,species such as L. donovani, and specially L.infantum, have a slow rate ofdivision (Gupta and Shakya, 2011) and some Leishmania isolates fromclinical cases do not infect the macrophage cell lines employed. By itspart, promastigotes are easy to culture and they share with amastigotesmany metabolic pathways and thus some of the most commonlyused antileishmanial agents, such as AmB or miltefosine, are effectiveagainst both parasite stages. Therefore, promastigote screening is use-ful (Sharlow et al., 2009) before testing in intracellular amastigotes,and in vivo, and can be employed to monitor susceptibility of clinicalisolates to some drugs (i.e. Miltefosine, Kulshrestha et al., 2013).
Among the available methods (3H-thymidine incorporation, mi-croscopic counting, quantitative PCR, enzymatic and colorimetricmethods) to determine proliferation of Leishmania promastigotes,resazurin-based assays such as Alamar Blue can be carried out in asingle step, are unaffected by the red phenol from the culture mediumand have the advantage of low cost and negligible toxicity. Validationwith L.donovani and L.infantum promastigotes showed that this methodcould be advantageously used in trials of cell proliferation and drug
screening, including the determination of approximate IC50 valuesagainst promastigotes. Conditions described for Leishmania includedthe presence of 5% CO2, or reduced air availability, to allow the efficientreduction of resazurin to resorufin, in a promastigote-density relatedmanner. The method was improved by fluorimetry although spectro-photometry could also be employed. Given the accuracy (fluorescencevs. promastigotes density), the low number of promastigotes neededand results obtained in the determination of IC50 for AmB and allicin ascompared to the results with microscopic counting (i.e. Corral-Caridadet al., 2012), this 96-well microplate could be adapted for a 384-wellformat.
Acknowledgements
Wedeeply thank thefinancial support byComisión Interministerial deCiencia y Tecnología [CICYT Grant (AGL2009-13009)]. MJC has a predoc-toral fellowship from the Ministerio de Ciencia e Innovación (MICINN).Excellent technical help by Mrs. Beatriz Rojas is acknowledged.
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