OMIP-079
Cell cycle of CD4+ and CD8+ naïve/memory T cell subsets, and of Treg cells from mouse spleen
TBD
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
MACHINE: BD LSR Fortessa
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 |