Blasticidin s hcl

All experiments were performed with rat basophilic leukaemia cells (RBL-703/21-derived NFATp-DsRed-Express2) generated at The University of Nottingham^{16 (link)}. These are available from the authors for non-commercial purposes on the basis of a material transfer agreement (MTA). Cells were grown in Minimum Essential Medium Eagle’s (EMEM) cell culture medium supplemented with 10% v/v heat-inactivated foetal bovine serum (Gibco, UK), 100 U/mL penicillin, 100 μg/mL streptomycin and 2 mM L-glutamine (Merck, UK) in a 37 °C/5% CO₂ humidified cell incubator. To maintain stable expression of FcεRIα_H, cells were cultured with 1 mg/mL G418 sulphate (Thermo Fisher Scientific, UK) and 20 μg/mL Blasticidin S HCL (InvivoGen, USA) for selection of NFATp-DsRed-Express2 reporter gene expression.

Parasites were cultured in vitro in complete RPMI medium containing Albumax II (lipid-rich bovine serum albumin) (Gibco) (0.5% w/v), gentamycin (Pfizer) (10 µg/ml), O + RBCs to 4% haematocrit (Australian Red Cross). Cultures were gassed with 1% O₂, 5% CO₂ and 94% N₂ (1 s/ml of culture) and kept closed airtight at 37 °C. Where required, cells were cultured under drug selection with either 5 nM WR99210 (Jacobus Pharmaceuticals) or 5.4 µM Blasticidin S-HCl InvivoGen).

SIRT1 shRNA (sc‐40986‐sh, Santa Cruz, United States) was used to generate SIRT1‐depleted hBMECs following the manufacturer's protocol. shRNA (1 µg) and shRNA Plasmid Transfection Reagent (sc‐108061, Santa Cruz, United States) were diluted in plasmid transfection medium (sc‐108062, Santa Cruz, United States), gently added onto cultured cells, and incubated for 12 h at 37 °C. Following incubation, a fresh medium was added, and the cells were incubated for an additional 24–48 h. To select SIRT1‐depleted cells, 5 µg mL⁻¹ puromycin dihydrochloride (ant‐pr‐1, InvivoGen, United States) was used.
SIRT1 CRISPR activation plasmid (sc‐400085‐ACT, Santa Cruz, United States) was used to generate SIRT1‐activated hBMECs following the manufacturer's protocol. Briefly, 1 µg of plasmid DNA and UltraCruz Transfection Reagent (sc‐395739, Santa Cruz, United States) were diluted in a plasmid transfection medium (sc‐108062, Santa Cruz, United States). Plasmid DNA/UltraCruz Transfection Reagent complex was added dropwise to cultured cells and incubated for 24–72 h. To select stably transfected cells, 5 µg mL⁻¹ puromycin dihydrochloride (ant‐pr‐1, InvivoGen, United States), 200 µg mL⁻¹ Hygromycin B (ant‐hg‐1, InvivoGen, United States), and 10 µg mL⁻¹ Blasticidin S HCl (ant‐bl‐05, InvivoGen, United States) were used.