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Optimization of the harvesting and freezing conditions of human cell lines for DNA damage analysis by the alkaline Comet assay

2018-12-10Journal article Restricted access
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dc.contributor.authorBessa, Maria João
dc.contributor.authorBrandão, Fátima
dc.contributor.authorMachado Querido, Micaela
dc.contributor.authorCosta Pereira, Cristiana
dc.contributor.authorValdiglesias, Vanessa
dc.contributor.authorLaffon, Blanca
dc.contributor.authorCarriere, Marie
dc.contributor.authorTeixeira, João Paulo
dc.contributor.authorFraga, Sónia
dc.date.accessioned2019-03-06T15:46:32Z
dc.date.available2019-03-06T15:46:32Z
dc.date.issued2018-12-10
dc.description.abstractThe comet assay is a commonly used method for in vitro and in vivo genotoxicity assessment. This versatile assay can be performed in a wide range of tissues and cell types. Although most of the studies use samples immediately processed after collection, frozen biological samples can also be used. The present study aimed to optimize a collection and freezing protocol to minimize the DNA damage associated with these procedures in human cell line samples for comet assay analysis. This study was conducted in glial A172 and lung alveolar epithelial A549 cells. Two cell detachment methods (mechanical vs enzymatic) and two cryoprotective media [FBS + 10% DMSO vs Cell Culture Media (CCM) + 10% DMSO] were tested, and DNA damage assessed at four time points following storage at −80 °C (one, two, four and eight weeks). In both cell lines, no differences in % tail intensity were detected between fresh and frozen cells up to eight weeks, irrespective of the harvesting method and freezing medium used. However, freshly isolated A172 cells exhibited a significant lower DNA damage when resuspended in CCM + 10% DMSO, while for A549 fresh cells the preferable harvesting method was the enzymatic one since it induced less DNA damage. Although both harvesting methods and cryoprotective media tested were found suitable, our data indicate that enzymatic harvesting and cryopreservation in CCM + 10% DMSO is a preferable method for DNA integrity preservation of human cell line samples for comet assay analysis. Our data also suggest that CCM is a preferable and cost-effective alternative to FBS in cryopreservation media. This optimized protocol allows the analysis of in vitro cell samples collected and frozen at different locations, with minimal interference on the basal DNA strand break levels in samples kept frozen up to eight weeks.pt_PT
dc.description.sponsorshipThis work was supported by the Portuguese Foundation for Scienceand Technology (FCT) through the CERASAFE project (SIINN/0004/2014). MJ. Bessa, F. Brandão and M.M. Querido thank FCT for their PhD scholarships (SFRH/BD/120646/2016, SFRH/BD/101060/2014 and SFRH/BD/130203/2017, respectively). V. Valdiglesias was sup-ported by a Xunta de Galicia Postdoctoral fellowship (ED481B 2016/190-0). The authors would also like to acknowledge the contribution ofthe hCOMET CA15132 COST Action.pt_PT
dc.description.versioninfo:eu-repo/semantics/publishedVersionpt_PT
dc.identifier.citationMutat Res Gen Tox En. 2018 Dec 10:1-7. doi: 10.1016/j.mrgentox.2018.12.002pt_PT
dc.identifier.doi10.1016/j.mrgentox.2018.12.002pt_PT
dc.identifier.urihttp://hdl.handle.net/10400.18/6075
dc.language.isoengpt_PT
dc.peerreviewedyespt_PT
dc.publisherElsevierpt_PT
dc.relation.publisherversionhttps://www.sciencedirect.com/science/article/pii/S1383571818303140pt_PT
dc.subjectDNA Damagept_PT
dc.subjectIn vitropt_PT
dc.subjectComet Assaypt_PT
dc.subjectA172 Cellspt_PT
dc.subjectA549 Cellspt_PT
dc.subjectCell Collectionpt_PT
dc.subjectCryopreservation Mediapt_PT
dc.subjectGenotoxicidade Ambientalpt_PT
dc.titleOptimization of the harvesting and freezing conditions of human cell lines for DNA damage analysis by the alkaline Comet assaypt_PT
dc.typejournal article
dspace.entity.typePublication
oaire.citation.endPage7pt_PT
oaire.citation.startPage1pt_PT
oaire.citation.titleMutation Research - Genetic Toxicology and Environmental Mutagenesispt_PT
rcaap.rightsembargoedAccesspt_PT
rcaap.typearticlept_PT

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