Automated multiparameter tissue based imaging cytometry
Current approaches of standard flow cytometry allow the analysis of about fifty parameters, however only in single cell suspension, thus losing all spatial information in the oftentimes crucial tissue context. Traditional immunohistochemistry lets researchers analyze just a few parameters due to spectral overlap of available reagents, any attempts at manual multiplexing approaches are tedious and time consuming. Imaging mass cytometry approaches overcome this hurdle, but they are limited by the number of suitable metal-isotopes for antibody tagging and are complex to operate.
The latest cutting edge ultra-high parameter imaging cytometry technology combines the use of standard fluorescent imaging and analysis of hundreds of proteins (and RNA) markers on the same tissue section for deep multiomic profiling and biomarker discovery.
Our core facility offers access to the Miltenyi MACSIma platform for fully automated measurement of a de facto unlimited number of markers using traditional immunohistochemistry in a cyclical process of staining, imaging and signal erasing. The process is known as MICS (MACSima Imaging Cyclic Staining).
Per cycle three antibodies tagged with one each of FITC (Alexa488 or similar), PE and APC (Alexa647) can be used to stain FFPE or fresh-frozen sections in situ with DAPI acting as nuclear stain. Tissue sections are mounted on so called MACSWell imaging frames designed for the complete MICS process. Expert histology support for optimal sectioning and QC is available from our colleagues at CF-PTA.
Ready made staining panels on dried-down antibody plates as well as custom/self-made panels of liquid antibodies can be used. Specific antibody reagents like the Miltenyi REAffnity antibodies (recombinant, low background due to lack of FcR binding site), REAlease antibodies (releases whole antibody-fluorochrome complex) or the Miltenyi REAdye_lease antibodies (releases just the fluorophore) can be used together with any suitable antibody from any other source for maximal flexibility. Signal emission is triggered by high power LEDs and captured via fluorescence microscopy in the regions of interest. The fluorescent signal is then erased by photobleaching and/or chemical release of the fluorophore (for REAdye_lease antibodies) before starting a new cycle of antibody staining. Tissues stay intact during the staining process and can be used in downstream applications.
Staining on the Miltenyi MACSima can also be combined with detection of RNA on the same tissue section for multiomics spatial biology. Data are captured on board the MACSima instrument, on the fly analysis is possible with the MACS iQView software.
After loading antibodies, tissues and buffers the instrument allows complete walk-away multiomics analysis of hundreds of markers. It overcomes typical multiplexing challenges such as manual workflows, a lack of readily available validated antibodies, and the need for robust data analysis software. Expert support in experimental design and training is available from our teams at CF-FLOW and CF-PTA.
For more information, please refer to this webinar on MICS technology and the Miltenyi Biotech webpage on the MACSima platform. To start an experiment on the Miltenyi MACSima platform please book a consultation session with CF-FLOW. Detailed experimental planning is crucial for the success of complex staining panels, please allow sufficient time for panel design, tissue preparation and reagent ordering.
Current approaches of standard flow cytometry allow the analysis of about fifty parameters, however only in single cell suspension, thus losing all spatial information in the oftentimes crucial tissue context. Traditional immunohistochemistry lets researchers analyze just a few parameters due to spectral overlap of available reagents, any attempts at manual multiplexing approaches are tedious and time consuming. Imaging mass cytometry approaches overcome this hurdle, but they are limited by the number of suitable metal-isotopes for antibody tagging and are complex to operate.
The latest cutting edge ultra-high parameter imaging cytometry technology combines the use of standard fluorescent imaging and analysis of hundreds of proteins (and RNA) markers on the same tissue section for deep multiomic profiling and biomarker discovery.
Our core facility offers access to the Miltenyi MACSIma platform for fully automated measurement of a de facto unlimited number of markers using traditional immunohistochemistry in a cyclical process of staining, imaging and signal erasing. The process is known as MICS (MACSima Imaging Cyclic Staining).
Per cycle three antibodies tagged with one each of FITC (Alexa488 or similar), PE and APC (Alexa647) can be used to stain FFPE or fresh-frozen sections in situ with DAPI acting as nuclear stain. Tissue sections are mounted on so called MACSWell imaging frames designed for the complete MICS process. Expert histology support for optimal sectioning and QC is available from our colleagues at CF-PTA.
Ready made staining panels on dried-down antibody plates as well as custom/self-made panels of liquid antibodies can be used. Specific antibody reagents like the Miltenyi REAffnity antibodies (recombinant, low background due to lack of FcR binding site), REAlease antibodies (releases whole antibody-fluorochrome complex) or the Miltenyi REAdye_lease antibodies (releases just the fluorophore) can be used together with any suitable antibody from any other source for maximal flexibility. Signal emission is triggered by high power LEDs and captured via fluorescence microscopy in the regions of interest. The fluorescent signal is then erased by photobleaching and/or chemical release of the fluorophore (for REAdye_lease antibodies) before starting a new cycle of antibody staining. Tissues stay intact during the staining process and can be used in downstream applications.
Staining on the Miltenyi MACSima can also be combined with detection of RNA on the same tissue section for multiomics spatial biology. Data are captured on board the MACSima instrument, on the fly analysis is possible with the MACS iQView software.
After loading antibodies, tissues and buffers the instrument allows complete walk-away multiomics analysis of hundreds of markers. It overcomes typical multiplexing challenges such as manual workflows, a lack of readily available validated antibodies, and the need for robust data analysis software. Expert support in experimental design and training is available from our teams at CF-FLOW and CF-PTA.
For more information, please refer to this webinar on MICS technology and the Miltenyi Biotech webpage on the MACSima platform. To start an experiment on the Miltenyi MACSima platform please book a consultation session with CF-FLOW. Detailed experimental planning is crucial for the success of complex staining panels, please allow sufficient time for panel design, tissue preparation and reagent ordering.