Isolation and flow cytometric characterization of protoplasts from marine macroalgae

Protoplasts were isolated from Ulva rigida C. Agardh (Chlorophyta) and two species of Rhodophyta, Gracilariopsis lemaneiformis (Bory) Dawson, Acleto et Folvik and Gracilaria tenuistipitata Chang et Xia var. liui with minor modifications (the inclusion of 0.01% agarase in the set of cell-wall-degrading enzymes for the two red algae). Flow cytometric characteristics of freshly isolated protoplasts were determined on a FACScan flow cytometer (FC). The most useful parameters for characterizing protoplasts from marine algae were forward angle light scatter (FSC), orange fluorescence (FL2) and red fluorescence (FL3). Protoplasts from all the species were easily distinguishable when their FSC, FL2, and FL3 signals were combined in the bivariate Plots FL3 vs. FSC and FL3 vs. FL2. Two alternative techniques to help identify protoplasts from debris in the FC computer screen were developed (for FC without sorting capability). Both techniques were based on the ability of new FCs to record time. The first one was based on the induction of rapid changes of cell volume in response to osmotic stress. Only intact protoplasts responded to changes in the osmotic pressure. The second one was based on the uptake and hydrolysis of fluorescein diacetate by intracellular esterases. Viable protoplasts showed a hyperbolic accumulation of fluorescein with time. Semimaximal fluorescein accumulation was attained in 30.5 +/- 9.5 s. Debris was easily recognized since, contrary to protoplasts, it did not show a time-dependent accumulation of fluorescein.