Lipofectamine rnai reagent

Cells were transfected with GATA3 siRNA using Lipofectamine RNAi reagent (Thermo Fisher Scientific). The sequences of GATA3 siRNA were: siRNA-1: 5′-GGCUCUACUACAAGCUUCACAAUAU-3′ and siRNA-2: 5′-CCACACUCUGGAGGAGGAAUGCCAA-3′.22 (link)

Femurs were flushed out with PBS using a sterile needle, and BMDM cells were cultivated in DMEM medium (Life Technologies, Carlsbad, CA, USA) containing L929 conditioned media. After 5 days, BMDMs cells were then incubated at 37°C in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin solution (Trouplin et al., 2013 ). Orai1 and TRPC1 adenovirus was purchased from AbmGood (Richmond, BC, Canada), and was amplified in HEK 293 using the manufacture's protocol. For overexpression experiments, 5 MOI of the virus encoding Orai1 or TRPC1 was added to 75% confluent cells. For silencing experiments respective Orai1siRNA (ThermoFisher, Assay ID: s99509) and TRPC1siRNA (ThermoFisher, Assay ID:104,443) was used, and the transfection was performed with Lipofectamine RNAi Reagent following the manufacture's protocol. Cells were seeded to be 70% confluent, and the Lipofectamine 2000 reagent was diluted in an optimal concentration (ThermoFisher, LMRNA015, 25 pmol per well in a 6-well dish) in Opti-MeM medium (Gibco, Dublin, Ireland). We added the lipofectamine to the diluted siRNA in a 1:1 ratio and incubated for 5 min at room temperature, after the stipulated time the complex was layered on the cells for 5 h followed by adding fresh media before using for individual experiments.