Poly4053

Cells were harvested using trypsin and stained with anti-myoferlin 7D2 (Abcam, ab76746), anti-CD151 conjugated to Allophycocyanin (Biolegend, #350405), or anti-CD99 conjugated to phycoerythrin (Biolegend, #318008) according to the manufacture’s recommendations. Secondary antibodies were used when appropriate (Biolegend, #Poly4053). Cells were stained for 40 minutes at 4 C followed by once the addition of 3 milliliters of PBS, centrifugation at 1000 × g for five minutes and suspension in PBS prior to flow cytometry on a BD FACS Canto Flow Cytometer. Data analysis was performed using FlowJo (Tree Star, Inc, Ashland, OR)

Serum was collected at various time points post-MHV-68 infection for the assessment viral-specific serum antibody as has been previously described [24 (link)]. Briefly, NIH/3T3 cells (ATCC CRL-1658) were infected with 0.03–0.1 MOI of MHV-68 and incubated at 37°C for 48 hours. Subsequently, monolayers were washed with PBS and subjected to mechanical disruption to allow for the collection of MHV-68 infected cell suspensions. Cell suspensions were then sonicated twice for 30 seconds to obtain a homogenous cell lysate. Cellular lysate was diluted 1:10 in sterile carbonate-bicarbonate buffer (Invitrogen) and 100ul per well was used to coat 96-well flat bottom microtiter plates (Immulon 4HBX, VWR). Plates were incubated for 48 hours at room temperature. A standard ELISA was then performed utilizing serum dilutions of 1:50 in blocking buffer and goat anti-mouse IgG-HRP (Poly4053, Biolegend) was used for the detection of MHV-68-specific immunoglobulins. Colorimetric development was performed using the TMB substrate system (Thermo Scientific) and acquired at 450nm on a Spectra MAX 340PC Microplate reader (Molecular Devices).