Recurrent rhinovirus infections in a child with inherited MDA5 deficiency

Lamborn, Ian T.; Jing, Huie; Zhang, Yu; Drutman, Scott B.; Abbott, Jordan K.; Munir, Shirin; Bade, Sangeeta; Murdock, Heardley M.; Santos, Celia P.; Brock, Linda G.; Masutani, Evan; Fordjour, Emmanuel Y.; McElwee, Joshua J.; Hughes, Jason D.; Nichols, Dave P.; Belkadi, Aziz; Oler, Andrew J.; Happel, Corinne S.; Matthews, Helen F.; Abel, Laurent; Collins, Peter L.; Subbarao, Kanta; Gelfand, Erwin W.; Ciancanelli, Michael J.; Casanova, Jean-Laurent; Su, Helen C.

At 24 and 48 h after RSV-GFP infection, whole well contents were collected for flow cytometric analysis. First, cell supernatants followed by sequential PBS washes were collected in 5 ml FACS tubes (Falcon). Next, single-cell suspensions were collected after treating with 0.25% trypsin/EDTA (Gibco) for 10 min and combining with cell supernatants and washes. Contents were washed 1 time with PBS and stained with LIVE/DEAD Fixable Near-IR Dead Cell Stain (Molecular Probes) or Zombie NIR Fixable Viability kit (BioLegend) for 20 min at room temperature, fixed in BD Cytofix/Cytoperm solution (BD) or 1% paraformaldehyde (Electron Microscopy Sciences) in PBS for 20 min, washed, and resuspended in PBS containing 1% FCS or 1% BSA and 0.09% sodium azide. The stained and fixed cells were acquired on a BD FACS Canto II or BD LSR II flow cytometers without compensation controls given the negligible spectral overlap between fluorophores. FlowJo 9.8.3 or 10.0.8 (Tree Star Inc.) software was used to enumerate singlet live cells expressing GFP. The values of % GFP + cells treated with siRNA to MDA5 are shown relative to the values for negative control siRNA.