Heat inactivated newborn calf serum

Primary Human Umbilical Vein Endothelial Cells (HUVECs) were isolated from umbilical cords of healthy donors. Umbilical cords were provided by the Amstelland Ziekenhuis, Amstelveen. Informed consents were obtained from all donors in accordance with the institutional guidelines and the Declaration of Helsinki. The cells were isolated and characterized as described by Jaffe et al.^{33 (link)}. The primary HUVECs were cultured in M199 medium supplemented with: penicillin 100 U/mL and streptomycin 100 μg/mL, L-glutamine 2 mMol/L (all from Bio Whittaker/Lonza), heat-inactivated human serum 10% (Sanquin, Amsterdam, The Netherlands), heat-inactivated new-born calf serum 10% (Gibco), crude endothelial cell growth factor 150 μg/mL (locally prepared from bovine brains) and heparin 5 U/mL (Leo pharmaceutical products, Weesp, The Netherlands). Cells were cultured at 37 °C and 5% CO₂, and medium was refreshed every second day. For all experiments, pools of HUVECs of 3 donors in passages 1–2 were used.

PBMC were isolated from SHIV89.6-infected and healthy macaques by Ficoll-hypaque centrifugation (GE Healthcare Life Sciences, Pittsburgh, PA, USA) and maintained in RPMI-1640 medium supplemented with 10% heat-inactivated newborn calf serum (Gibco, Shanghai, China). Prior to use, cells were activated by incubating PBMC in complete medium with 10 µg/mL concanavalin A (Con A) and 50 U/mL recombinant IL-2. Cells were kept at 37 °C for 72 h in a humidified incubator with 5% CO₂ for activation.

For all liquid filamentation assays, saturated overnight cultures were diluted to an optical density at 600 nm (OD₆₀₀) of 0.1 in the indicated media. Heat-inactivated newborn calf serum (catalog no. 26010074; Gibco) was used at 10% in RPMI 1640 medium with l-glutamine and sodium bicarbonate (catalog no. R8758; Sigma) with 5% CO₂. For N-acetylglucosamine (GlcNAc), 5 mM was prepared in YNB medium (1× YNB, 2% Casamino Acids, 1× uridine, and 0.2% glucose). Spider medium and Lee’s medium were prepared as previously described (5 (link)). For all liquid media, strains were grown under shaking conditions. Filamentation was assessed at 5 h under all conditions. For solid cues, saturated overnight cultures were diluted 10-fold in water, and 5 μL was spotted onto the appropriate agar plates. Filamentation was assessed at 72 h. All strains were grown at 37°C except for the elevated temperature cue for which strains were grown in YPD medium at 39°C. Cell morphology for liquid conditions was captured using differential interference contrast (DIC) microscopy on a Zeiss Axio Imager.M1 (Carl Zeiss). Colony morphology for solid conditions was captured using a Bio-Rad ChemiDoc imaging system. Images are representative of two biological replicates.