A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes
| Autor: | Soizic Garaud, Anaïs Boisson, Hugues Duvillier, Jean Nicolas Lodewyckx, Pushpamali De Silva, Céline Naveaux, Laurence Buisseret, Karen Willard-Gallo, Myriam Libin, Chunyan Gu-Trantien, Edoardo Migliori |
|---|---|
| Rok vydání: | 2014 |
| Předmět: |
Chemokine
Pathology medicine.medical_specialty General Chemical Engineering Receptor expression Cytological Techniques Immunology Breast Neoplasms General Biochemistry Genetics and Molecular Biology Lymphocytes Tumor-Infiltrating Immune system medicine Humans Breast General Immunology and Microbiology biology Tumor-infiltrating lymphocytes General Neuroscience In vitro Cell biology Tumor Escape Tumor progression biology.protein Leukocyte Common Antigens Female Antibody |
| Zdroj: | Journal of Visualized Experiments. |
| ISSN: | 1940-087X |
| Popis: | The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45(+) cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45(+) cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues. |
| Databáze: | OpenAIRE |
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