The CellSensor® STAT6-bla RA-1 human B-cell line from Invitrogen (Life Technologies, Grand Island, NY, USA) was used. Cells were propagated in Opti-MEM Reduced Serum Medium containing 5% heat-inactivated fetal bovine serum, 100 μm nonessential amino acids, 1 mm sodium pyruvate, 1% penicillin/streptomycin, and 556 ng/mL of CD40 Ligand (Invitrogen, Life Technologies) overnight at 37 °C, 5% CO2. Cells were then plated in 384-well assay plates, black-wall, clear bottom (Costar Corning, Corning, NY, USA) at a density of 20 000 cells per well. Oclacitinib (0.0000381–10 μm ) or vehicle control was added to cells for 2 h. Ten nanograms per milliliter hIL-4 (Invitrogen) was then added to cell cultures for 5 h. Activation of the STAT6-beta-lactamase reporter gene by IL-4 was determined by detecting beta-lactamase activity with the LiveBLAzer™-FRET B/G substrate (CCF-4 AM; Invitrogen) for 2 h. Fluorescence emission values at 460 and 530 nm were obtained using a fluorescent plate reader. The 460/530 nm ratios were expressed as percent control, and dose–response data were analyzed using a 4-parameter logistic equation.
Opti mem reduced serum
Manufactured by Thermo Fisher Scientific
Opti-MEM reduced serum is a cell culture medium designed to support cell growth and transfection while reducing the amount of serum required. It is formulated to maintain cell health and support various cell types in reduced serum conditions.
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Lab products found in correlation
Rnaimax, by Thermo Fisher Scientific (2 mentions)
Liveblazer fret b g substrate ccf 4 am, by Thermo Fisher Scientific (1 mentions)
Lipofectamine ltx and plus reagent, by Thermo Fisher Scientific (1 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (1 mentions)
Gamma counter, by Hidex (1 mentions)
Lowry based protein assay, by Bio-Rad (1 mentions)
Gsc 6201g, by Tebu-Bio (1 mentions)
Smart pool on targetplus sirnas, by Horizon Discovery (1 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (1 mentions)
6 protocols using opti mem reduced serum
1
STAT6-driven IL-4 Activation Assay
2
H9c2 Cardiomyocyte Pcsk9 Silencing and Overexpression
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H9c2 cells (CRL-1446, America Tissue Type Collection) were differentiated into H9c2 cardiomyocytes as described in the Supplementary Material . For Pcsk9 silencing experiments, H9c2 cardiomyocytes were transfected with rat PCSK9-−specific siRNA and control siRNA (20 µmol/L, Horizon Discovery) in distilled water and Lipofectamine RNAiMax transfection reagent (Invitrogen). For Pcsk9 overexpression experiments, H9c2 cardiomyocytes were transfected with PCSK9-GFP cDNA (1 µg) or empty control CT-GFP (Sinobiological) in Optimem Reduced Serum and Lipofectamine LTX and Plus Reagent (Invitrogen).
3
Manganese Uptake Kinetics Assay
4
Transient Transfection of Mouse Embryonic Stem Cells
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Mouse R1 embryonic stem cells (ESCs) were obtained from Sigma-Aldrich (07072001-1VL) and cultivated on a layer of gamma-irradiated CF-1 mouse embryonic fibroblasts (Tebu-Bio, GSC-6201G) in DMEM formulated with high glucose and sodium pyruvate and supplemented with 15% FBS, 103 units/mL LIF, 1% β-mercaptoethanol, 100 units/mL penicillin, 100 μg/mL streptomycin and maintained at 37 °C and 5% CO2 in a humidified incubator. For transfection, 2 × 105 ESCs were incubated with 800 ng of plasmid for 15 min in suspension42 (link). Imaging was done within 24–48 h following transfection. In the starvation experiment, the medium was replaced by OptiMEM reduced serum (Thermo Fisher Scientific, 31985-062).
5
Knockdown studies in EndoC-βH1 cells
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Knockdown studies in EndoC-βH1 cells were performed using Lipofectamine RNAiMAX® transfection protocol and 15 nM SMART pool ON-TARGETplus siRNAs (Horizon Discovery Biosciences, siNT: D-001810-10-05, siPAX4: L-012240-00-0005) diluted in Opti-MEM reduced serum-free medium (ThermoFisher Scientific, 31985062) and 0.4% RNAiMAX® (ThermoFisher Scientific, 13778150). Silencing efficiency was determined by qPCR from samples collected during GSIS, five days post-transfection.
6
siRNA Knockdown of DAXX
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All siRNA transfections were performed by using Lipofectamine RNAi Max reagent (13778075; Thermo Fisher Scientific) according to the manufacturer’s instructions. For RNAi, siRNA oligos and transfecting reagent were diluted in Opti-MEM reduced serum media (31985088; Thermo Fisher Scientific). siRNA oligo against DAXX (5′-CTGGAACCTGGCAAACAGAT-3′) was ordered from Dharmacon (Shrestha et al., 2017a (link)). For control siRNA, negative control oligo (12935-300; Invitrogen) was used.
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