Cryopreservation is commonly used for storing viable cells, tissues, organs or organisms at ultralow temperatures, and usually involves immersion in liquid nitrogen at -196 °C. Here we provide a detailed cryopreservation protocol for C. reinhardtii based on Crutchfield’s work (Crutchfield et al., 1999), with minor changes (Yang and Li, 2016). In this study, we compared the cryoprotection effect of two common cryopreservation agents (CPAs), methanol and DMSO. Furthermore, the two-step cryopreservation process was divided into five stages to study the factors affecting the survival rate at each stage. We found that the use of methanol as the CPA, combined with the cooling process outlined here (cooling from 25 °C to -55 °C at a rate of 1 °C/min), were indispensable for cell survival after cryopreservation. The thawing process described here (thawing at 35 °C for 5 min) was also important for increasing the survival rate.
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