Cryopreservation has consistently proven to be a viable method for the storage of a wide variety of biological material, and there has been a recent focus on the cryopreservation of Symbiodinium spp. given the role of these dinoflagellates in the biology of the coral hosts with which they regularly associate. The present study aimed to identify the effect of various cryoprotectants (CPAs) on clade G Symbiodinium isolated from the whip coral Junceella fragilis by analyzing (i) dinoflagellate cell membrane integrity (via SYTOX® staining) and (ii) metabolic function (via an ATP assay) after cryopreservation. At low concentrations (1 M), none of the CPAs tested were harmful to the dinoflagellates at up to 60 min of incubation, and methanol and DMSO were the least harmful; neither caused a significant effect on cell ATP content even after 30 min incubations at 4 M concentrations. Detrimental effects of the CPAs increased in the following order: MeOH (lowest) = DMSO < EG < PG < Gly. With respect to the different assays, the ATP bioassay was more sensitive to CPA exposure than SYTOX staining. Given these findings, MeOH and DMSO should see more widespread use in cryopreservation protocols for clade G Symbiodinium, as well as other dinoflagellates.

中文翻译：

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使用光度计和流式细胞术评估冷冻保护剂在柳珊瑚内共生体 Symbiodinium 中的作用

冷冻保存一直被证明是储存各种生物材料的可行方法，并且最近重点关注共生菌属的冷冻保存。考虑到这些甲藻在它们经常与之交往的珊瑚宿主的生物学中的作用。本研究旨在通过分析 (i) 甲藻细胞膜完整性（通过 SYTOX® 染色）和 (ii) 代谢功能（通过 ATP化验）后冷冻保存。在低浓度 (1 M) 下，所测试的 CPA 在长达 60 分钟的孵育时间均对鞭毛藻无害，甲醇和 DMSO 的危害最小；即使在 4 M 浓度下孵育 30 分钟后，也不会对细胞 ATP 含量产生显着影响。CPA 的有害影响按以下顺序增加：MeOH（最低）= DMSO < EG < PG < Gly。对于不同的检测，ATP 生物检测对 CPA 暴露比 SYTOX 染色更敏感。鉴于这些发现，MeOH 和 DMSO 应该会在进化枝 G Symbiodinium 以及其他甲藻的冷冻保存方案中得到更广泛的应用。