Bacterial growth and invasion into HUVECs

Bacteria (B. henselae WT, transposants 623-125, and 804-29, OD₆₀₀ = 0.05, 100 µL) were plated on CSB agar plates to evaluate the bacterial growth. After the indicated days, bacteria were harvested from the agar surface by extensive washing with 1 mL of PBS. The OD₆₀₀ of the bacterial suspensions were measured in a JASCO V-630BIO spectropolarimeter (JASCO Corp.).

A gentamicin protection assay was used to calculate the number of intracellular bacteria. HUVECs seeded in a 24-well plate were infected with B. henselae WT or 623-125 (MOI = 200) for 24 h at 37 °C in a humidified atmosphere and 5% CO₂. After the infection, gentamicin sulfate (200 µg/mL) was added for 2 h to kill extracellular bacteria. The cells were then washed with Medium 199 (M199, Gibco) supplemented with 10% FBS three times and lysed by incubation for 15 min with 1% saponin. Cell lysates including intracellular bacteria were resuspended with 1000-fold volume of PBS. The numbers of bacteria were determined by plating on CSB agar.