Biomarker discovery efforts in translational medicine are increasingly reliant on antibody-mediated approaches for protein detection in both suspension and fixed tissue sections. Using the Helios™ CyTOF system and Hyperion™ Imaging System, the Houston Methodist Immunomonitoring Core will facilitate immune phenotyping – a technique to study the protein expressed by cells – and spatial analysis of the tumor microenvironment. The imaging system enables simultaneous interrogation of 4 to 37 protein markers using proven CyTOF® technology together with imaging capability.
The use of highly pure metal labels on antibodies in place of fluorescent tags provides a solution to the current challenges in multiplexed tissue imaging by separating signals based on differences in mass instead of wavelength to overcome the limitations of fluorescence-based detection modalities.
The Hyperion Imaging System makes it possible to deeply interrogate tissues and tumors at subcellular resolution while preserving the information in tissue architecture and cellular morphology, ideal for characterization of the tissue microenvironment across a breadth of disease research areas. The Helios systems focuses on suspension samples at cellular level to reveal the whole profile of cellular components within a tumor/disease environment.
For use of the Helios™ CyTOF system, depending on the tissue type, it is preferred that tissue be processed and digested into single cell suspension with one to three million cells per sample. Red blood cells and dead cells should be removed, otherwise samples will be returned since dead cells will aggregate and clog the Helios™ CyTOF system. Samples must be dropped off on ice before 3 p.m. on the scheduled day.
For Imaging Mass Cytometry, tissue should be fixed by formalin or PFA for 24 hours or longer depending on the tissue type. When arranging or embedding tissue in a cassette, tissue should be placed in the middle of the cassette so the section will not be at the edge of the slide. If the tissue has a long shape, like bone marrow, the tissue should be placed horizontally or vertically, without any angles, leaving no gaps between the tissue so the laser does not hit the glass.
The fixed tissue may be embedded in paraffin. After conjugating antibodies of interest (if applicable) and finalizing the antibody panel, the embedded tissue will then be sent to sectioning, since fresh epitope on the tissue is preferred. Tissue should be sectioned in 5um with only one section on each slide. One H&E slide and four unconjugated slides should be provided. Please image the H&E slides in low magnification (1X or 2X) and provide the images with the circled area to ablate.
For the Helios™ CyTOF system, cells will be stained with metal-conjugated viability dye and block. Surface markers will be stained first before fixation. If there are intracellular markers in the panel, cells will be fixed with Foxp3 staining kit and stained with intracellular antibodies. Cells will be incubated in metal-conjugated cell ID overnight at 4 degrees C. Samples will be filtered through a 40um strainer before acquisition.
For Imaging Mass Cytometry, slides will be incubated at 60 degrees C overnight to allow better tissue attachment and depariffination. Slides will be depariffinated and rehydrated by three times xylene and serial-concentration ethanol. Antigen-retrieval will use Tris-EDTA and will be incubated in a temperature-control microwave at 95 degrees C for 20 minutes (if another antigen retrieval buffer and protocol is preferred, the method can be adjusted for a specific tissue type). Slides will be chilled and blocked with 3% BSA for one hour. Slides will be incubated with metal-conjugated antibodies at 4 degrees C overnight. The next day, the slides will be washed and stained with metal-labelled cell ID for the nuclei. Slides will be kept at 4 degrees C until sample acquisition.
TeamShu-Hsia Chen, PhD
Emily Herrmann Chair in Immunology Research, Cancer Center
Director, Center for Immunotherapy Research
Director, Immunomonitoring Core
Jilu Zhang, PhD
Immune Assessment Core Manager
Yitian Xu, PhD
Immune Assessment Core Operator
Immune Assessment Core Technician