Compensation beads

ECs and HUVECs were plated on 6 well plates. Six h later, stably mCherry-expressing U251T3 cells (U251T3-mCherry) were collected and infected with 0.1 MOI rHSVQ or OVesRAGE virus for 30 min in a suspension condition. Cells were washed and overlaid on top of an equivalent number of ECs and cultured for 24 h. Cells were then collected and stained with Live/Dead Fixable Aqua Dead Cell Stain Kit (Life Technologies, Eugene, OR, USA) and analyzed in CytoFlex (Beckman Coulter, Brea, CA, USA). Single-stain controls for each fluorochrome were prepared using cells or compensation beads (Invitrogen, cat #01-222-42, Waltham, MA, USA) for compensation. Tumor and ECs were initially separated by gating for the mCherry-positive population (550–650 nm emission) and then further gated for GFP expression and live/dead cell staining-positive population. GFP expression represents virus-infected cells, while live/dead staining represents dead cell population. Data were analyzed using FlowJo v.10.7.

Cells were detached and washed once with 1x PBS and resuspended in FACs buffer (0.5% BSA, 2 mM EDTA in PBS). Staining with extracellular fluorochrome coupled-antibodies was carried out in 100uL of FACs buffer for 20 minutes in the dark at 4 °C. Antibodies used for macrophage phenotyping were: CD80 (BV605 BD Biosciences), CD83 (BV650, BD Biosciences), CD86 (BV786, BD Biosciences), CD200R (PE, Biolegend), CD206 (Alexa-Fluor647, BD Biosciences), CD209 (BV421, BD Biosciences). Viability staining was performed by adding one drop of Sytox green flow reagent (ThermoFisher Scientific) to samples prior to analysis. Compensation was performed using either Ultracomp eBeads plus compensation beads (Invitrogen). All samples were rewashed in FACs buffer once before being analyzed using a BD LSR-Fortessa X20 cell analyzer. Data were analyzed using the FlowJo software (BD Biosciences).

Cells were collected in the same manner as described in the Protein Extraction section. For each sample, 4 × 10⁵–1 × 10⁶ cells were stained. First, live cells were identified by adding 1 μL of eBioscience Fixable Viability Dye was added per 1 × 10⁶ cells for 30 minutes at 4°C in the dark. Cells were then centrifuged at 500xg at room temperature for 5 minutes, washed with FACS buffer (PBS containing 0.05% azide and 2% FBS), then stained with 5 μL of phycoerythrin (PE) mouse anti-human E-cadherin antibody (BD Biosciences, 562870) per 1 × 10⁶ cells for 30 minutes at 4°C. Cells were washed twice, then fixed and permeabilized using eBioscience IC Fixation Buffer and Permeabilization Buffers (Thermo Fischer Scientific, 88-8824-00) as described by the manufacturer. Cells were stained with 5 μL of Alexa 488 mouse anti-human vimentin antibody (BD Biosciences, 562338) per 1 × 10⁶ cells for 20 minutes at room temperature. Samples were washed once with 1X Permeabilization Buffer and once with FACS buffer. Finally, cells were resuspended in FACS buffer and analyzed on a BD LSRII flow cytometry (BD Biosciences). As a staining control, compensation beads (eBioscience, Thermo Fisher Scientific, 01-2222-41) were also stained with PE mouse anti-human E-cadherin stain and Alexa 488 mouse anti-human vimentin stain, and fixed following staining. Gating strategy is shown in Supplementary Figure 1.