Cell cycle of CD4+ and CD8+ naïve/memory T cell subsets, and of Treg cells from mouse spleen

Ambra Natalini,Sonia Simonetti,Gabriele Favaretto,Giovanna Peruzzi,Fabrizio Antonangeli,Angela Santoni,Miguel Muñoz-Ruiz,Adrian Hayday,Francesca Di Rosa

PURPOSE: A multicolor flow cytometry panel was designed and optimized to define the following nine mouse T cell subsets: Treg (CD3+ CD4+ CD8− FoxP3+), CD4+ T naïve (CD3+ CD4+ CD8−FoxP3− CD44int/low CD62L+), CD4+ T central memory (CD3+ CD4+ CD8− FoxP3− CD44high CD62L+), CD4+ T effector memory (CD3+ CD4+ CD8− FoxP3− CD44high CD62L−), CD4+ T EMRA (CD3+ CD4+ CD8− FoxP3− CD44int/low CD62L−), CD8+ T naïve (CD3+ CD8+ CD4− CD44int/low CD62L+), CD8+ T central memory (CD3+ CD8+ CD4− CD44high CD62L+), CD8+ T effector memory (CD3+ CD8+ CD4− CD44high CD62L−), and CD8+ T EMRA (CD3+ CD8+ CD4− CD44int/low CD62L−). In each T cell subset, a dual staining for Ki-67 expression and DNA content was employed to distinguish the following cell cycle phases: G0 (Ki67−, with 2n DNA), G1 (Ki67+, with 2n DNA), and S-G2/M (Ki67+, with 2n < DNA ≤ 4n). This panel was established for the analysis of mouse (C57BL/6J) spleen.

CELL TYPE: Splenocytes


Marker Target Clone Fluorophore Purpose
CD3 Mouse 145-2C11 FITC Pan T cell marker
CD44 Mouse IM7 APC NaÏve/Memory subset identification
Ki-67 Mouse SolA15 Alexa Fluor 700 Quiescence/cell cycle
eFluor 780 Fix Viability All Species eFluor 780 Fix Viability Live/Dead cell discrimination
Hoechst (33342) All Species Hoechst (33342) DNA content/cell cycle
FoxP3 Mouse FJK-16s PE Treg identification
CD4 Mouse RM4-5 PE-CF594 Helper T cell identification
CD62L Mouse MEL-14 PE-Cy7 NaÏve/Memory subset identification
CD8 Mouse 53-6.7 Brilliant Violet 785 Cytotoxic T cell identification
CD16/CD32 Mouse 2.4G2 Purified