Optimization of the cryopreservation of biological resources, Toxoplasma gondii tachyzoites, using flow cytometry.

Alexandre Mzabi; Sandie Escotte-Binet; Richard Le Naour; Naïma Ortis; Sandra Audonnet; Marie-Laure Dardé; Dominique Aubert; Isabelle Villena

Optimization of the cryopreservation of biological resources, Toxoplasma gondii tachyzoites, using flow cytometry.

Alexandre Mzabi ^* Sandie Escotte-Binet Richard Le Naour Naïma Ortis Sandra Audonnet Marie-Laure Dardé ^{1, 2, 3} Dominique Aubert Isabelle Villena

* Auteur correspondant

1 NET - Neuroépidémiologie Tropicale

2 UNILIM - Université de Limoges

3 CNR Toxoplasmose-Toxoplasma BRC - Centre National de Référence (CNR) Toxoplasmose/Toxoplasma Biological Resource Center (BRC)

Abstract : The conservation of Toxoplasma gondii strains isolated from humans and animals is essential for conducting studies on Toxoplasma. Conservation is the main function of the French Biological Toxoplasma Resource Centre (BRC Toxoplasma, France, http://www.toxocrb.com/). In this study, we have determined the suitability of a standard cryopreservation methodology for different Toxoplasma strains using the viability of tachyzoites assayed by flow cytometry with dual fluorescent labelling (calcein acetoxymethyl ester and propidium iodide) of tachyzoites. This method provides a comparative quantitative assessment of viability after thawing. The results helped to define and refine quality criteria before tachyzoite cryopreservation and optimization of the cryopreservation parameters. The optimized cryopreservation method uses a volume of 1.0 mL containing 8 × 10(6) tachyzoites, in Iscove's Modified Dulbecco's Medium (IMDM) containing 10% foetal calf serum (FCS). The cryoprotectant additive is 10% v/v Me2SO without incubation. A cooling rate of ∼1 °C/min to -80 °C followed, after 48 h, by storage in liquid nitrogen. Thawing was performed using a 37 °C water bath that produced a warming rate of ∼100 °C/min, and samples were then diluted 1:5 in IMDM with 5% FCS, and centrifuged and resuspended for viability assessment.

Type de document :

Article dans une revue

Domaine :

Sciences du Vivant [q-bio] / Santé publique et épidémiologie

Liste complète des métadonnées

https://hal-unilim.archives-ouvertes.fr/hal-01262835
Contributeur : Elisabeth Grelier <>
Soumis le : mercredi 27 janvier 2016 - 11:37:41
Dernière modification le : jeudi 20 septembre 2018 - 16:22:01

Optimization of the cryopreservation of biological resources, Toxoplasma gondii tachyzoites, using flow cytometry.

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Optimization of the cryopreservation of biological resources, Toxoplasma gondii tachyzoites, using flow cytometry.

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