ACME-sorbitol (ACMEsorb) Fixation and Enzymatic Dissociation of Whole Soft-Bodied Cnidarians v1

ACME (Acetic-Methanol) maceration is a tissue dissociation and fixation method described by Jordi Solana’s group that preserves both cell morphology and RNA integrity, and is compatible with single-cell transcriptomics (scRNA-seq) (García-Castro et al., 2021). ACME has been tested on various organisms, including freshwater planarians (Dugesia japonica), annelids (Pristina leidyi), snails (Limnaea stagnalis), vertebrate embryos (Mus musculus and Danio rerio), terrestrial arthropods (Parasteatoda tepidariorum embryos and Drosophila melanogaster larvae), and the starlet sea anemone (Nematostella vectensis), which inhabits brackish waters (García-Castro et al., 2021). However, for marine organisms, this protocol proved suboptimal in our hands, as we observed cells bursting immediately upon contact with the fixative solution—presumably due to the hypo-osmolarity of the fixative relative to seawater. To compensate for the osmolarity and preserve cell integrity during fixation, we replaced the water (or 1× PBS) fraction in the original ACME formulation with a 0.8 M sorbitol solution (Najle et al., 2023). This modified version, which we named ACMEsorb, was successfully applied for single-cell atlasing of various marine species. ACMEsorb fixation of whole organisms, followed by cell dissociation, is the preferred method, as it minimizes cellular stress and reduces viability biases observed during the dissociation of fresh samples. Dissociation post-fixation can be performed either enzymatically, as described here, or mechanically (a protocol using mechanical dissociation after ACMEsorb fixation is available [here]). In this protocol, we use cold-protease to dissociate whole specimens of the starlet sea anemone N. vectensis, after a short fixation with ACMEsorb.