Computational protocol: Rhinovirus stimulated IFN-α production: how important are plasmacytoid DCs, monocytes and endosomal pH?

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Protocol publication

[…] Intracellular cytokine staining was used to assess IFN-α-producing pDCs, monocytes and mDCs after RV16 (multiplicity of infection of 1) stimulation. PBMCs (2 × 106) per well were seeded in a 96-well U-bottom plate and stimulated with or without RV16 at 37 °C with 5% CO2 for 18 h, and further incubated with Brefeldin A (eBioscience, San Diego, CA, USA) for 4 h. Cells were washed with FACs buffer (1% heat-inactivated fetal calf serum in phosphate-buffered saline; fetal calf serum; Bovogen Biologicals, Keilor East, VIC, Australia) and incubated with normal goat IgG (Sigma-Aldrich) at 4 °C for 15 min to block non-specific Fc binding. The cells then were surface stained with CD303-PE, CD14-PerCP and CD1c-FITC (Miltenyi Biotec, Bergisch Gladbach, Germany) for 30 min at 4 °C, then fixed and permeabilized before antigen-presenting cell conjugated anti-IFN-α (Miltenyi Biotec) intracellular staining for 30 min at 4 °C. The cells were then washed twice with the FACs buffer, finally fixed in 0.5% paraformaldehyde before analysis. A total of ~500 000 gated events per sample were collected using FACS Canto (BD Biosciences, Franklin Lakes, NJ, USA), and the results were analyzed using the FlowJo Tree Star software (version 7.6.1; Flowjo LLC, Ashland, OR, USA). Unstimulated background was subtracted from the data. [...] Data were analyzed in IBM SPSS Statistics and GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA) using Friedman tests with Dunn's post-tests to compare paired samples, whereas Mann–Whitney test was used to compare data from asthmatic and healthy subjects. Raw data are presented as mean±s.e.m. The and represent the median and interquartile ranges. The P-values <0.05 were considered significant. […]

Pipeline specifications

Software tools FlowJo, SPSS
Applications Miscellaneous, Flow cytometry
Organisms Homo sapiens
Diseases Asthma, Infection, Virus Diseases
Chemicals Chloroquine