Many critical advances in research utilize techniques that combine high-resolution with high-content characterization at the single cell level. We introduce the MICS (MACSima Imaging Cyclic Staining) technology, which enables the immunofluorescent imaging of hundreds of protein targets across a single specimen at subcellular resolution. MICS is based on cycles of staining, imaging, and erasure, using photobleaching of fluorescent labels of recombinant antibodies (REAfinity Antibodies), or release of antibodies (REAlease Antibodies) or their labels (REAdye_lease Antibodies). Multimarker analysis can identify potential targets for immune therapy against solid tumors. With MICS we analysed human glioblastoma, ovarian and pancreatic carcinoma, and 16 healthy tissues, identifying the pair EPCAM/THY1 as a potential target for chimeric antigen receptor (CAR) T cell therapy for ovarian carcinoma. Using an Adapter CAR T cell approach, we show selective killing of cells only if both markers are expressed. MICS represents a new high-content microscopy methodology widely applicable for personalized medicine.

Background: Use of microfluidics in point-of-care testing (POCT) will require on-board fluidics, self-contained reagents, and multistep reactions, all at a low cost. Disposable microchips were studied as a potential POCT platform. Methods: Micron-sized structures and capillaries were embedded in disposable plastics with mechanisms for fluidic control, metering, specimen application, separation, and mixing of nanoliter to microliter volumes. Designs allowed dry reagents to be on separate substrates and liquid reagents to be added. Control of surface energy to ؎5 dyne/cm 2 and mechanical tolerances to <1 m were used to control flow propulsion into adsorptive, chromatographic, and capillary zones. Fluidic mechanisms were combined into working examples for urinalysis, blood glucose, and hemoglobin A 1c testing using indicators (substances that react with analyte, such as dyes, enzyme substrates, and diazonium salts), catalytic reactions, and antibodies as recognition components. Optical signal generation characterized fluid flow and allowed detection. Results: We produced chips that included capillary geometries from 10 to 200 m with geometries for stopping and starting the flow of blood, urine, or buffer; vented chambers for metering and splitting 100 nL to 30 L; specimen inlets for bubble-free specimen entry and containment; capillary manifolds for mixing; microstructure interfaces for homogeneous transfer into separation membranes; miniaturized containers for liquid storage and release; and moisture vapor barrier seals for

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Many critical advances in research utilize techniques that combine high-resolution with high-content characterization at the single cell level. We introduce the MICS (MACSimaTM Imaging Cyclic Staining) technology, which enables the immunofluorescent imaging of hundreds of protein targets across a single specimen at sub-cellular resolution. MICS is based on cycles of staining, imaging, and erasure, using photobleaching of fluorescent labels of recombinant antibodies (REAfinityTM), release of antibodies (REAleaseTM) or their labels (REAdyeleaseTM). Multimarker analysis can identify potential targets for immune therapy against solid tumors. With MICS we analysed human glioblastoma, ovarian and pancreatic carcinoma, and 16 normal tissues. One potential target pair for chimeric antigen receptor (CAR) T-cell therapies identified for ovarian carcinoma is EPCAM/THY1. Using an adapter CAR T cell approach, we show selective killing of cells only in presence of both markers. MICS represents a new high content microscopy methodology to be widely used for personalized medicine.