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Cellular Encapsulation and Immunostaining Using a Photo Polymerized Hydrogel
Author(s) -
Din Shabana,
White Laura,
Saito Kan,
Desmond Lisa,
MilliporeSigma Jeffrey Turner
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.09887
Subject(s) - flow cytometry , staining , cell , jurkat cells , live cell imaging , multiplex , biomedical engineering , fluorescence microscope , chemistry , cytometry , microbiology and biotechnology , fluorescence , pathology , immune system , t cell , biology , immunology , medicine , bioinformatics , biochemistry , physics , quantum mechanics
Rationale Immunocytochemistry is a valuable technique used to visualize protein expression, localization, and cellular structural changes by microscopic imaging. However, immunofluorescence of suspension cells, such as primary immune cells or rare circulating tumor cells, can be technically challenging requiring either expensive equipment and or mechanical manipulation. Furthermore, low amount of cellular sample can prevent proper assessment of experimental results and reduce throughput. Objective Here we introduce a photo polymerized hydrogel, Cell Capture, which uniformly encapsulates non‐adherent cells and permits immuno labeling and visualization by fluorescence imaging. Methods and Results Cell Capture reagent was formulated and optimized for maximal permeability to antibodies while ensuring cellular retention within the hydrogel. Preparation of samples for immunoassaying in microtiter plates led to decreased workflow times, compared to antibody staining in bulk processing due to the removal of centrifugation steps. Immortalized T lymphocyte Jurkat cells were plated in varying cell numbers, ranging from 4×10 5 to 5×10 3 , followed by staining with CD3 antibody. Cell Capture demonstrated minimal cell loss with phenotypic membrane staining of CD3 and additional T cell targets, including multiplex tri color detection by microscopy. Cell Capture was further compared to traditional method of non‐adherent cell immobilization by extra cellular matrix coating of surfaces. Superior cellular retention properties were observed when using Cell Capture, demonstrating the advantages of this technology. Image cytometry is a common method to image suspension cells but requires a significant financial investment. To demonstrate Cell Capture as an alternative to image cytometry, cells were fixed into Cell Capture hydrogel following flow cytometry evaluation. Imaging by fluorescence microscopy, showed clear and distinct staining of measured immune targets in Jurkat cells, verifying Cell Capture as a valid alternative to image cytometry. Conclusion Cell Capture reagent allows for convenient immune labeling and detection of nonadherent cells in a high throughput fashion, demonstrating the utility and flexibility over traditional suspension cell immunocytochemistry methods.