UMDNJ-Robert Wood Johnson Medical School

Described here are protocols used to visualize the dynamic process of MG53-mediated cell membrane repair in whole animals and at the cellular level. These methods can be applied to investigate the cell biology of plasma membrane resealing and regenerative medicine.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

We describe a method of measuring binding energy, expressible as tissue surface tension, between cells within 3D tissue-like aggregates. Differences in tissue surface tension have been demonstrated to correlate with invasiveness of lung, muscle, and brain tumors, and are fundamental determinants of establishing spatial relationships between different cell types.

The extent of store-operated Ca²⁺ entry (SOCE) can be monitored using fluorescent Ca²⁺ indicators. Mn²⁺ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca²⁺ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

Described here is a method to directly measure calcium sparks, the elementary units of Ca²⁺ release from sarcoplasmic reticulum in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca²⁺ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca²⁺ signaling can be employed to assess muscle function in health and disease.

Here we report common methods to analyze the phagocytic function of murine alveolar macrophages and bacterial clearance from the lung. These methods study in vitro phagocytosis of fluorescein isothiocyanate beads and in vivo phagocytosis of Pseudomonas aeruginosa Green Fluorescent Protein. We also describe a method for clearing P. aeruginosa in mice.

Here, we describe a protocol for detection and localization of Drosophila embryo protein and RNA from collection to pre-embedding and embedding, immunostaining, and mRNA in situ hybridization.