Coral cell separation and isolation by fluorescence-activated cell sorting (FACS)

Abstract

BackgroundGeneralized methods for understanding the cell biology of non-model species are quite rare, yet very much needed. In order to address this issue, we have modified a technique traditionally used in the biomedical field for ecological and evolutionary research. Fluorescent activated cell sorting (FACS) is often used for sorting and identifying cell populations. In this study, we developed a method to identify and isolate different cell populations in corals and other cnidarians.MethodsUsing fluorescence-activated cell sorting (FACS), coral cell suspension were sorted into different cellular populations using fluorescent cell markers that are non-species specific. Over 30 different cell markers were tested. Additionally, cell suspension from Aiptasia pallida was also tested, and a phagocytosis test was done as a downstream functional assay.ResultsWe found that 24 of the screened markers positively labeled coral cells and 16 differentiated cell sub-populations. We identified 12 different cellular sub-populations using three markers, and found that each sub-population is primarily homogeneous. Lastly, we verified this technique in a sea anemone, Aiptasia pallida, and found that with minor modifications, a similar gating strategy can be successfully applied. Additionally, within A. pallida, we show elevated phagocytosis of sorted cells based on an immune associated marker.ConclusionsIn this study, we successfully adapted FACS for isolating coral cell populations and conclude that this technique is translatable for future use in other species. This technique has the potential to be used for different types of studies on the cellular stress response and other immunological studies.Electronic supplementary materialThe online version of this article (10.1186/s12860-017-0146-8) contains supplementary material, which is available to authorized users.

Novel methods to establish whole-body primary cell cultures for the cnidarians Nematostella vectensis and Pocillopora damicornis

“…Cnidarians, and in particular scleractinian corals, are also critically important for ocean biodiversity and health 8 , 9 . However, much about the cell biology of anthozoans is still unknown 10 , 11 , in part due to there being no reliable cell culture method established 12 – 14 .…”

Lineage dynamics of the endosymbiotic cell type in the soft coral Xenia

“…Eight polyps (about 10 8 cells) were fixed with 4% paraformaldehyde (PFA) overnight. After washing twice with 3.3× PBS 38 and dissociating the tissue in 2 ml 3.3× PBS using a 7-ml glass Dounce tissue grinder (Wheaton), another 3 ml 3.3× PBS was added. The mixture was then transferred to a 15-ml conical tube and centrifuged at 1,000 g for 3 min (Sorvall Lynx 6000 centrifuge, ThermoFisher Scientific).…”

Dielectrophoresis Separation of Platelets Using a Novel Zigzag Microchannel

“…Microfluidics is a simple, low-cost, and easy-to-operate technology compared with traditional macroscale technology that requires large-scale operation equipment and a suitable laboratory facility. The principle of separation in microfluidic systems operates via well-designed microchannels and precisely controlled external physical separation methods that utilize acoustic fields [ 11 , 12 ], fluorescence activation [ 13 , 14 ], optical filing [ 15 , 16 ], magnetic activation [ 17 , 18 ], and electrical fields [ 19 , 20 , 21 ]. Shamloo et al [ 11 ] simulated the separation of blood cells with acoustic fields, while evaluating the most efficient and low-cost separation devices by altering factors of acoustic field devices.…”

“…Shamloo et al [ 11 ] simulated the separation of blood cells with acoustic fields, while evaluating the most efficient and low-cost separation devices by altering factors of acoustic field devices. In addition, Rosental et al [ 14 ] demonstrated and developed a method to isolate different cell populations in corals and other cnidarians using fluorescent activated cell sorting (FACS) technology, which is an advanced cell sorting technique. Further, Tamura et al [ 15 ] used optical cell separation technology to separate and classify heterogeneous cancerous cells based on morphology, confirming the feasibility of optical technology in cancer cell manipulation.…”