TY - JOUR T1 - Workflow for high-dimensional flow cytometry analysis of T cells from tumor metastases JF - Life Science Alliance JO - Life Sci. Alliance DO - 10.26508/lsa.202101316 VL - 5 IS - 10 SP - e202101316 AU - Cristina Faccani AU - Gianluca Rotta AU - Francesca Clemente AU - Maya Fedeli AU - Danilo Abbati AU - Francesco Manfredi AU - Alessia Potenza AU - Achille Anselmo AU - Federica Pedica AU - Guido Fiorentini AU - Chiara Villa AU - Maria P Protti AU - Claudio Doglioni AU - Luca Aldrighetti AU - Chiara Bonini AU - Giulia Casorati AU - Paolo Dellabona AU - Claudia de Lalla Y1 - 2022/10/01 UR - https://www.life-science-alliance.org/content/5/10/e202101316.abstract N2 - We describe a multi-step high-dimensional (HD) flow cytometry workflow for the deep phenotypic characterization of T cells infiltrating metastatic tumor lesions in the liver, particularly derived from colorectal cancer (CRC-LM). First, we applied a novel flow cytometer setting approach based on single positive cells rather than fluorescent beads, resulting in optimal sensitivity when compared with previously published protocols. Second, we set up a 26-color based antibody panel designed to assess the functional state of both conventional T-cell subsets and unconventional invariant natural killer T, mucosal associated invariant T, and gamma delta T (γδT)-cell populations, which are abundant in the liver. Third, the dissociation of the CRC-LM samples was accurately tuned to preserve both the viability and antigenic integrity of the stained cells. This combined procedure permitted the optimal capturing of the phenotypic complexity of T cells infiltrating CRC-LM. Hence, this study provides a robust tool for high-dimensional flow cytometry analysis of complex T-cell populations, which could be adapted to characterize other relevant pathological tissues. ER -