Microalgal cell division tracking using CFSE

icroalgal cell division tracking could unlock new research means. It could help decipher cell response to an intentional stressor, aiming at increasing an added value molecule accumulation, or an accidental stressor, in the case of environmental pollution. It could also be used to monitor asynchrony in cultures exposed to photoperiod or determine the fate of dead cells. To date, because of the lack of guidelines specific to microalgae, microalgal ecotoxicology and biotechnology communities have not yet implemented such protocol in routine. Therefore, a systematic optimization methodology has been deployed to adapt the CarboxyFluorescein DiAcetate Succinimidyl Ester (CF-DA-SE, or, in short, CFSE) lymphocytes proliferation tracking technique to the microalga Chlorella vulgaris. The toxic effect of the CFSE solvent (DMSO) was delineated (stock solution at 10 mM). Then, incubation conditions (time, probe/cell ratio, illumination) were optimized (30 min, 4.50 nmol/MCell, in the dark). Finally, CFSE washing and cell recovery were robustified (low-acceleration - 100 g - centrifugation). Using a semi-synchronous culture as a test case, the method was successfully applied to count cell divisions. Up to four generations could be discriminated. The generation-to-generation signal ratio was exactly 1/4, corresponding to the natural division of Chlorella vulgaris. Furthermore, an advanced yet easy-to-implement signal processing technique was introduced to ease generation discrimination.