Computational protocol: Human β-D-3 Exacerbates MDA5 but Suppresses TLR3 Responses to the Viral Molecular Pattern Mimic Polyinosinic:Polycytidylic Acid

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Protocol publication

[…] BMDM (2 x 104 cells/well) were cultured overnight on 8 well glass chamber slides (Nunc Inc, IL, USA) in DMEM with 10% FCS. Cells were treated with FITC-polyI:C (2.5μg/ml) in Optimem media (Life Technologies) with or without lipofectamine 2000 (Invitrogen, at 1:100 dilution, 10μl/ml) in the presence or absence of hBD3 or TAMRA-labelled HBD3 (0.5μg/ml). After 2, 10, 15 or 30 mins cells were washed in PBS and fixed in 4% PFA. For early endosome staining, cells were blocked with 10% donkey serum and incubated with anti-EEA11 antibody (Abcam, UK) for 1 hr at RT, then 30 min with donkey anti-rabbit Cy5. Cells were imaged using a 40x 1.3NA oil immersion objective on a Nikon A1R confocal microscope using Nikon Nis-Elements AR software for image acquisition (Nikon Instruments Europe, Netherlands). Image analysis was carried out in ImageJ (http://imagej.nih.gov/ij/). Pearsons coefficients were calculated using the JaCoP ImageJ plugin []. […]

Pipeline specifications

Software tools ImageJ, JACoP
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus, Homo sapiens
Chemicals Poly I, Poly I-C