To determine whether coating of VV was successful, flow virometry of VV coated with Chol-PEG(10K)-FITC (named FITC-PCVV) was performed.
YOYO-3 (Thermo Fisher Scientific, Y3606) was used for nucleic acid staining of VV.13 (link),44 VV was incubated with 1 μM YOYO-3 at room temperature for 20 min. The sample was then centrifuged at 20,000 × g for 10 min. The viral pellet was washed three times in PBS as described in section 4.1. Final virus pellets were either resuspended in PBS to a concentration of 5.3 × 105 virus particles (VG)/μL and further modified to form PCVV, or 2.6 × 105 VG/μL as the naked form.44 Stained VV virions further modified to PCVV were coated and purified as detailed in section 4.1, and resuspended at a final concentration of 2.6 × 105 VG/μL. An Attune NxT cytometer (Invitrogen) was adapted to record FSC and SSC through the violet channel using the Attune NxT No-Wash No-Lyse filter kit (Thermo Fisher Scientific, 100022776). VV populations were gated for positive YOYO-3 staining. Analysis was performed using FlowJo v10.6.1 software. FITC-PCVV-YOYO-3 and VV-YOYO-3 were gated for positive YOYO-3 and FITC staining.
YOYO-3 (Thermo Fisher Scientific, Y3606) was used for nucleic acid staining of VV.13 (link),44 VV was incubated with 1 μM YOYO-3 at room temperature for 20 min. The sample was then centrifuged at 20,000 × g for 10 min. The viral pellet was washed three times in PBS as described in section 4.1. Final virus pellets were either resuspended in PBS to a concentration of 5.3 × 105 virus particles (VG)/μL and further modified to form PCVV, or 2.6 × 105 VG/μL as the naked form.44 Stained VV virions further modified to PCVV were coated and purified as detailed in section 4.1, and resuspended at a final concentration of 2.6 × 105 VG/μL. An Attune NxT cytometer (Invitrogen) was adapted to record FSC and SSC through the violet channel using the Attune NxT No-Wash No-Lyse filter kit (Thermo Fisher Scientific, 100022776). VV populations were gated for positive YOYO-3 staining. Analysis was performed using FlowJo v10.6.1 software. FITC-PCVV-YOYO-3 and VV-YOYO-3 were gated for positive YOYO-3 and FITC staining.
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