Lentiviral Knockdown of Dectin-1 in BMDMs

Lentivirus generation and transduction of shRNA was performed as described previously [23 (link)]. The lentiviral construct vector pLKO.1 and three packaging plasmids (pMDLg/pRRE, pRSV-Rev and pMD2.VSV-G) were from Open Biosystems (Huntsville, AL, USA) and target dectin-1 (TRCN0000066928) was from Sigma-Aldrich, supplied as glycerol stocks. Lentivirus generation was achieved by transfecting with Lipofectamine 2000 into HEK 293 T cells with pLKO puro.1 or target shRNA plasmid and the packaging plasmids. After 72 h, the virus-containing HEK 293 T cell culture supernatants were collected and filtered. Lentivirus determination was performed as described previously [23 (link)]. The lentivirus particles mixed with 8 μg/mL Polybrene (Sigma-Aldrich) and non-specific shRNA or dectin-1 shRNA into BMDMs, according to the manufacturer’s protocol. After transduction, the BMDMs were harvested and the target gene-silencing efficiency was examined by RT-PCR analysis.