Endothelial growth medium

Primary human SC were obtained from iXCells Biotechnologies (catalog number 10HU-149) and were cultured using a 1:1 Dulbecco’s modified Eagle’s medium (DMEM) to F-12 medium supplemented with HEPES, L-glutamine, 100 U/mL penicillin-streptomycin, and 5% fetal bovine serum (FBS) as described previously (Siemann et al., 2017 (link)). A549 cells were cultured using DMEM supplemented with 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U/mL penicillin-streptomycin, 1X non-essential amino acids, 10 mM HEPES, and 10% fetal bovine serum (FBS). HBMVEC obtained from Cell Systems Corporation (catalog number CSC-2M1) were cultured using Endothelial Growth Medium (Angio-Proteomie, catalog number cAP-02). ZIKV strain PRVABC59 (Human/2015/Puerto Rico), acquired from American Type Culture Collection (ATCC), was propagated once in Vero E6 cells for virus stock preparation. ZIKV infection experiments were conducted by exposing cells to the virus at a multiplicity of infection (MOI) of 1 or 3 for 1 h at 37°C and 5% CO₂. The cells were subsequently washed with phosphate buffered solution (PBS) prior to the addition of fresh media. ZIKV progeny in cell supernatant and intracellular ZIKV RNA was quantified by plaque assay and RT-qPCR, respectively, as reported previously (Verma et al., 2009 (link); Adams Waldorf et al., 2016 (link)).

C166 murine yolk-sac endothelial cells (ATCC) were propagated in flasks coated with 0.1% gelatin (Stem Cell Technologies) using endothelial growth medium (Angioproteomie) and incubated at 37 °C and 5% CO₂ atmosphere. Monocytes were isolated from bone marrow of male CX3CR1^GFP/+ mice and sorted using a FACS-Aria IIIu to discriminate CX3CR1^hi monocytes and CX3CR1^lo monocytes. Endothelial tube forming assays were performed in 15-well angiogenesis slides (Ibidi) as follows. Wells were coated with BD Matrigel^TM and seeded with C166 cells (7500 cells/well) and CX3CR1^hi or CX3CR1^lo monocytes (2000 cells/well). After 20 hours, co-cultures were fixed and labeled with rhodamine-phalloidin. Images were captured using a Zeiss LSM 700 confocal (10x magnification) and Zen software (Zeiss). The centroid of each monocyte was identified and the distance to the nearest endothelial tube formation was calculated using a custom MATLAB code. For each vessel image, a random distribution of “cells” was overlaid on the image (equal to the number of monocytes in the original image) and the distance of the “random cells” to the nearest vessel was calculated. The experimental monocyte distance to vessel was then compared to the computer-generated randomized cell distance to vessel to determine whether the cells preferentially distribute themselves near vessel segments.