Preparing Planarian Cells for High-content Fluorescence Microscopy Using RNA in situ Hybridization and Immunocytochemistry

High-content fluorescence microscopy combines the efficiency of high-throughput techniques with the ability to extract quantitative information from biological systems. The planarian community has developed sensitive and robust assays for whole animals, yet cell based assays, despite their practical aspects, have not been explored to the same extent. Here we describe a modular collection of detailed protocols adapted for fixed planarian cells that enable multiplexed measurements of biomarkers in microwell plates. Methods include the detection of RNA transcripts by RNA fluorescent in situ hybridization combined with tyramide signal amplification using hapten-labeled riboprobes. In addition, immunocytochemical protocols for quantifying proliferating cells by the detection of phosphorylated histone H3 as well as 5-bromo-2'-deoxyuridine incorporation into the nuclear genome are described. The assays are compatible with planarians of virtually any size, as the tissue is disaggregated into a single cell suspension before fixation and staining. By sharing many reagents with established planarian whole mount staining protocols, preparation of samples for high-content microscopy adoption requires little additional investment. Recommendations for successful experimental workflows and common sources of errors are discussed.

Preparation of Single Cell Suspension from Human Lung Tissue v1

This protocol describes a method for the isolation of the immune cells, structural and epithelial cells, and progenitors from human lung sections of about two grams. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples.

Preparation of Single Cell Suspension from Human Lymph Node Tissue v1

This protocol describes a method for the isolation of pan-lymphocytes, pan-myeloid cells, and progenitors from human lymph node tissue. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples.

Preparation of Single Cell Suspension from Human Spleen Tissue v1

This protocol describes a method for the isolation of pan-lymphocytes, pan-myeloid cells, and progenitors from human spleen tissue. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples.

Preparation of a Single Cell Suspension from Bronchoalevolar Lavage v1

This protocol describes a method for the isolation of the immune cells, structural and epithelial cells, and progenitors from lavage fluid collected from human lung. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples.

Biomaterials for Cell-Surface Engineering and Their Efficacy

Literature in the field of stem cell therapy indicates that, when stem cells in a state of single-cell suspension are injected systemically, they show poor in vivo survival, while such cells show robust cell survival and regeneration activity when transplanted in the state of being attached on a biomaterial surface. Although an attachment-deprived state induces anoikis, when cell-surface engineering technology was adopted for stem cells in a single-cell suspension state, cell survival and regenerative activity dramatically improved. The biochemical signal coming from ECM (extracellular matrix) molecules activates the cell survival signal transduction pathway and prevents anoikis. According to the target disease, various therapeutic cells can be engineered to improve their survival and regenerative activity, and there are several types of biomaterials available for cell-surface engineering. In this review, biomaterial types and application strategies for cell-surface engineering are presented along with their expected efficacy.

Single cell digestion of tumor tissue v1

The purpose of this protocol is to provide details on how to obtain a single-cell suspension from a patient-derived xenograft tumor.