Rabbit anti ev d68 vp1

Cell suspensions of SK-N-SH or RD cells were incubated with EV-D68 strains for 1 h at 4°C using an MOI of 1. As a negative control, viruses were heat inactivated at 62°C for 10 min before incubation with the cells. Subsequently, cells were washed with PBS, fixed with 4% PFA for 15 min, and blocked for 30 min with PBS containing 5% normal goat serum (Dako, Denmark). Cells were incubated with rabbit anti-EV-D68 VP1 (20 μg/ml; GeneTex) in 2 mM EDTA (Sigma-Aldrich) and 0.1% BSA (Aurion) fluorescence-activated cell sorting (FACS) buffer for 1 h at 4°C. After washing 3 times, cells were incubated with a secondary goat-anti rabbit IgG conjugated to Alexa Fluor 594 (10 μg/ml; Life Technologies) in FACS buffer. After incubation, cells were washed 3 times in FACS buffer and analyzed using a BD FACS Lyrics flow cytometer (BD Bioscience, USA). The percentage of cells to which virus attached was determined using FlowJo 10 software (Ashland, OR, USA). Experiments were performed at least 3 times, and each experiment was performed in duplicate.

SK-N-SH and RD cells (∼80% confluent) in a 96-well plate were infected with EV-D68 at an MOI of 2. Mock-infected cells were included as a negative control. EV-D68-infected SK-N-SH and RD cells were incubated at 37°C and 33°C in 5% CO₂, respectively. After 8 hpi, cells were fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature, washed with PBS, and permeabilized with 70% ethanol. Cells were first incubated with 5% bovine serum albumin (BSA; Aurion, Wageningen, The Netherlands) in PBS for 30 min before incubation with rabbit anti-EV-D68 VP1 (20 μg/ml; GeneTex, Irvine, CA, USA) for 1 h. Cells were washed twice with PBS and incubated with goat anti-rabbit IgG conjugated with Alexa Fluor 594 (10 μg/ml; Life Technologies, Inc., The Netherlands) in PBS with 0.1% BSA (Aurion) for 1 h. Cells were washed 3 times with PBS and mounted with ProLong Diamond Antifade with DAPI (4′,6-diamidino-2-phenylindole; Life Technologies) to visualize nuclei. Each experiment included negative and omission controls. EV-D68 VP1-positive cells were identified using a Zeiss LSM 700 laser scanning microscope. All images were processed using Zen 2010 software. Per sample, 3 high-power fields were photographed, and the number of infected cells was calculated by counting virus-infected/uninfected cells in 3 randomly chosen panels. All experiments were performed in triplicate.