Using multiple imaging modalities while performing independent experiments in parallel can greatly enhance the throughput of microscopy-based research, but requires provision of appropriate experimental conditions in a format that meets the microscopy’s optical requirements. Although customized imaging chambers can meet these challenges, the difficulty of manufacturing custom chambers and the relatively high cost and design inflexibility of commercial chambers has limited the adoption of this approach. Herein, we demonstrate the use of 3D printing to produce inexpensive, customized live-cell imaging chambers that are compatible with a range of imaging modalities including super-resolution microscopy. In this approach, biocompatible plastics are used to print imaging chambers designed to meet the specific needs of an experiment, followed by adhesion of the printed chamber to a glass coverslip, producing a chamber that is impermeant to liquids and which supports the growth and imaging of cells over multiple days. This approach can also be used to produce moulds for casting PDMS microfluidic devices. The utility of these chambers is demonstrated using designs for multiplex microscopy, imaging under shear, chemotaxis, and general cellular imaging. Together, this approach represents an inexpensive yet highly customizable approach to produce imaging chambers that are compatible with modern microscopy techniques.

RNA drug delivery by red blood cell extracellular vesicles OPEN ACCESS | www.cell-stress.com Please cite this article as: Chanh Tin Pham, Xin Zhang, Austin Lam, Minh TN Le (2018). Red blood cell extracellular vesicles as powerful carriers of RNA-based therapeutics. Cell Stress 2(9): 239-241.

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Apoptosis, the programmed and intentional death of senescent, damaged, or otherwise superfluous cells, is the natural end-point for most cells within multicellular organisms. Apoptotic cells are not inherently damaging, but if left unattended, they can lyse through secondary necrosis. The resulting release of intracellular contents drives inflammation in the surrounding tissue and can lead to autoimmunity. These negative consequences of secondary necrosis are avoided by efferocytosis—the phagocytic clearance of apoptotic cells. Efferocytosis is a product of both apoptotic cells and efferocyte mechanisms, which cooperate to ensure the rapid and complete removal of apoptotic cells. Herein, we review the processes used by apoptotic cells to ensure their timely removal, and the receptors, signaling, and cellular processes used by efferocytes for efferocytosis, with a focus on the receptors and signaling driving this process.

While new high-resolution microscopy techniques are continually developed, adoption of these methods is often difficult due to an inability to meet the experimental conditions required for an experiment in a format which also meets the demanding optical requirements of these microscopy techniques. Although specialized imaging chambers can meet these challenges, the difficulty of manufacturing customized chambers in-house and the relatively high cost and design inflexibility of commercial chambers has limited the incorporation of imaging chambers into fluorescence and super-resolution microscopy experiments. Herein, we demonstrate the use of fused deposition modeling (3D printing) for producing inexpensive, customized imaging chambers that are compatible with long-duration live-cell imaging using fluorescence and super-resolution microscopy techniques. In this approach, biocompatible 3D printing plastics are used to generate imaging chambers designed to meet the specific needs of an experiment, followed by adhesion of the printed chamber to a glass coverslip suitable for fluorescence and super-resolution imaging. This technique produces a chamber that is impermeant to liquids that can support the growth and imaging of cells over multiple days. The utility of these chambers is then demonstrated using designs for multiplex microscopy, imaging under shear, chemotaxis, and general cellular imaging. Together, this approach represents an inexpensive yet highly customizable approach to produce imaging chambers that are compatible with many modern microscopy techniques.

Efferocytosis – the phagocytic removal of apoptotic cells – is a central component of tissue homeostasis and contributes to inflammatory and autoimmune conditions including atherosclerosis and multiple sclerosis. MER tyrosine kinase (MERTK) is the predominant or sole efferocytic receptor in multiple tissues, and is the predominant efferocytic receptor on macrophages. MERTK is an opsonin-dependent receptor tyrosine kinase that triggers a poorly understood signaling pathway to mediate apoptotic cell engulfment. Using immunoprecipitation, mass spectrometry, and super-resolution microscopy, we have identified a pre-formed receptor complex on the macrophage plasma membrane comprised of ~100 nm clusters of MERTK, β2 integrins, and multiple signaling molecules including Src-family kinases, PI3-kinases, and the integrin regulatory proteins ILK and FAK. MERTK is unable to mediate efferocytosis in the absence of β2 integrins or their opsonins, while β2 integrins require activation via MERTK signaling to induce the engulfment of apoptotic cells. Using FRET microscopy, we determined that MERTK directly induces the conformational change of β2 integrins from the low to high-affinity form via a PI3-kinase, FAK and ILK-dependent signaling pathway. Activation of this pathway then drives the coalescence of the MERTK/β2 integrin clusters into a larger synapse through which efferocytosis occurs. The identification of the MERTK signaling pathway and the role of β2 integrins in this pathway provides new insights into the function of this critical homeostatic receptor and provides new insights into how MERTK mutations and signaling defects may contribute to inflammatory and autoimmune diseases.

Apoptosis, the programmed and intentional death of senescent, damaged, or otherwise superfluous cells, is the natural end-point for most cells within multicellular organisms. Apoptotic cells are not inherently damaging, but if left unattended they can lyse through secondary necrosis. The resulting release of intracellular contents drives inflammation in the surrounding tissue and can lead to autoimmunity. These negative consequences of secondary necrosis are avoided by efferocytosis—the phagocytic clearance of apoptotic cells. Efferocytosis is a product of both apoptotic cell and efferocyte mechanisms, which cooperate to ensure the rapid and complete removal of apoptotic cells. Herein, the processes used by apoptotic cells to ensure their timely removal, and the receptors, signaling, and cellular processes used by efferocytes to identify, remove, and process the apoptotic cells, are reviewed.