This protocol facilitates the identification and isolation of live coral cell populations at a descriptional level not previously possible. The main advantage of this technique is that it allows access to a level of cell specificity that up until now has been mostly unattainable. Demonstrating the procedure will be Dr.Shannon Saigh, a research associate from the flow cytometry shared resource at the Sylvester Comprehensive Cancer Center.
To set up the flow cytometer for live coral cell sorting, open a new project template in the flow cytometer software and select the appropriate lasers for the analysis in the laser panel. Then, create a side scatter versus forward scatter plot in the appropriate plots for setting up the gating and analysis strategy. For flow cytometric analysis and sorting of the live coral cells, load the control unstained cell sample into the sample chamber of the cytometer and start the reading process.
Since coral cells are particularly fragile, keep the cytometer pressure low to prevent cell lysis. In the scatter plot, adjust the photo multiplier voltage to center the points and begin recording the events. In the forward versus side scatter plot, create a gate around the cells at around the 10 to the 4th mark on the forward scatter x-axis.
Replace the control sample with the first sample of stained cells and record approximately 10, 000 cells before pausing. Then gate the events that are unique to the stained sample group. To sort live coral cells, after selecting the cell population of interest in the flow cytometer software, place one microcentrifuge tube containing 250 to 500 microliters of the appropriate collection solution into the cytometer collection chamber for each population to sort.
Once an appropriate amount of time has passed to flush out debris, begin sorting the desired population to collect anywhere from 10, 000 to several million cells. Each time a new stain, species, or sorter is used, sort at least 20, 000 cells of interest into 500 microliters of staining medium to allow a purity check to be performed on the population of interest and reanalyze the sorted cells to confirm that the events are being read within the gate used for the initial sorting. For in vitro studies, store the sorted cells on ice until they're transferred to an incubator or sterilized environment.
To bring in other non-cellular particles that do not share the same shape or granularity as the coral cells can be removed from the analysis by creating a gate selection on a forward and side scatter plot. The dead cells can be excluded by comparing the DAPI signal of the unstained and stained cell suspensions using a histogram and gating out the high DAPI-expressing cells. In parallel, cells hosting autofluorescence symbiodeneasia can be removed by gating against cells with the high signal in the far red channel.
After this iterative gating process, the remaining cells represent the intact live coral cell populations lacking symbiodeneasia. These cells can then be classified into different cell subpopulations by staining for features such as reactive oxygen species activity and/or lysosome content. It's critical to remember that some cells are more fragile than others and to run this process as carefully as possible.
Once specific cells have been isolated, they can be used for x-vivo functional assays such as establishing cell cultures and creating transcriptomic profiles.
