%0 Journal Article %A Cristina Faccani %A Gianluca Rotta %A Francesca Clemente %A Maya Fedeli %A Danilo Abbati %A Francesco Manfredi %A Alessia Potenza %A Achille Anselmo %A Federica Pedica %A Guido Fiorentini %A Chiara Villa %A Maria P Protti %A Claudio Doglioni %A Luca Aldrighetti %A Chiara Bonini %A Giulia Casorati %A Paolo Dellabona %A Claudia de Lalla %T Workflow for high-dimensional flow cytometry analysis of T cells from tumor metastases %D 2022 %R 10.26508/lsa.202101316 %J Life Science Alliance %P e202101316 %V 5 %N 10 %X 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. %U https://www.life-science-alliance.org/content/lsa/5/10/e202101316.full.pdf