Sirt1 Overexpression in Mouse B Cells

Sirt1 coding region cDNA was amplified from unstimulated mouse B cells using the appropriate primers (table S1) and cloned into the pMIG retroviral expression vector. To generate the retrovirus, the pMIG vector encoding green fluorescent protein (GFP) only or the pMIG-Sirt1 vector encoding GFP and Sirt1 was used with the pCL-Eco retrovirus-packaging vector (Imgenex) to transfect human embryonic kidney–293T cells by a Ca⁺⁺ phosphate (ProFection Mammalian Transfection System, Promega). Viral supernatants were collected and used to transduce spleen B cells from C57BL/6 mice after a 12-hour LPS activation, as we reported (6 (link)). Transduced B cells were then stimulated with LPS plus IL-4 for 96 hours before analysis of GFP⁺ and/or IgG1⁺ B cells by flow cytometry. Dead (7-AAD⁺) cells were excluded from analysis.