Copyright © 2021 JoVE Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License jove.com January 2021 • 167 • e61867 • Page 1 of 19 A DNA/Ki67-Based Flow Cytometry Assay for Cell Cycle Analysis of Antigen-Specific CD8 T Cells in Vaccinated Mice Sonia Simonetti *,1,2 , Ambra Natalini *,1,2 , Giovanna Peruzzi 3 , Alfredo Nicosia 4 , Antonella Folgori 5 , Stefania Capone 5 , Angela Santoni 2,6 , Francesca Di Rosa 1 1 Institute of Molecular Biology and Pathology, National Research Council of Italy (CNR) 2 Department of Molecular Medicine, University of Rome “Sapienza” 3 Center for Life Nano Science, Istituto Italiano di Tecnologia 4 Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II 5 Reithera Srl 6 IRCCS, Neuromed * These authors contributed equally Corresponding Author Francesca Di Rosa francesca.dirosa@cnr.it Citation Simonetti, S., Natalini, A., Peruzzi, G., Nicosia, A., Folgori, A., Capone, S., Santoni, A., Di Rosa, F. A DNA/Ki67- Based Flow Cytometry Assay for Cell Cycle Analysis of Antigen-Specific CD8 T Cells in Vaccinated Mice. J. Vis. Exp. (167), e61867, doi:10.3791/61867 (2021). Date Published January 5, 2021 DOI 10.3791/61867 URL jove.com/video/61867 Abstract The cell cycle of antigen-specific T cells in vivo has been examined by using a few methods, all of which possess some limitations. Bromodeoxyuridine (BrdU) marks cells that are in or recently completed S-phase, and carboxyfluorescein succinimidyl ester (CFSE) detects daughter cells after division. However, these dyes do not allow identification of the cell cycle phase at the time of analysis. An alternative approach is to exploit Ki67, a marker that is highly expressed by cells in all phases of the cell cycle except the quiescent phase G 0 . Unfortunately, Ki67 does not allow further differentiation as it does not separate cells in S-phase that are committed to mitosis from those in G 1 that can remain in this phase , proceed into cycling, or move into G 0 . Here, we describe a flow cytometric method for capturing a "snapshot" of T cells in different cell cycle phases in mouse secondary lymphoid organs. The method combines Ki67 and DNA staining with major histocompatibility complex (MHC)-peptide-multimer staining and an innovative gating strategy, allowing us to successfully differentiate between antigen-specific CD8 T cells in G 0 , in G 1 and in S-G 2 /M phases of the cell cycle in the spleen and draining lymph nodes of mice after vaccination with viral vectors carrying the model antigen gag of human immunodeficiency virus (HIV)-1. Critical steps of the method were the choice of the DNA dye and the gating strategy to increase the assay sensitivity and to include highly activated/proliferating antigen- specific T cells that would have been missed by current criteria of analysis. The DNA dye, Hoechst 33342, enabled us to obtain a high-quality discrimination of the G 0 / G 1 and G 2 /M DNA peaks, while preserving membrane and intracellular staining. The