Lucifer yellow

HCMEC/D3 cells were grown until confluent on 0.4 μm pore size MilliCell^® inserts in 24-well cell plates. Log-phase S. suis was incubated with hFg (20 μg/ml) for 1 h and then added to the hCMEC/D3 cell monolayers at a MOI of 100:1. At the same time, Lucifer yellow (200 μM, Invitrogen) was added to the apical chamber and incubated in transport buffer (EBM-2 medium supplemented with 10 mM HEPES and 1 mM sodium pyruvate) for 30 min. The amount of Lucifer yellow in the basolateral chamber was quantified with a spectrophotometer (Varioskan Flash, Thermo Fisher).

We determined the permeability of Caco-2 cell monolayers by measuring the transepithelial passage of a fluorescent dye, lucifer yellow (Invitrogen).^{(13 (link))} lucifer yellow is a fluorescein dye with a molecular weight of 444. Caco-2 cells (48,000 cells/well) were harvested on cell culture inserts. Cells were washed with Hank’s Balanced Salt Solution (HBSS, Invitrogen) twice. Then, HBSS (600 µl) was added to the basolateral side chamber and HBSS containing 100 µM lucifer yellow (450 µl) was applied to the apical side chamber. Then, the cultures were placed on a shaker in a 5% CO₂ incubator at 37°C to ensure adequate mixing. Samples (100 µl) of the basolateral chambers were analyzed for fluorescence using a fluorescence microplate reader (Tecan, Zurich, Switzerland) at an excitation wavelength of 485 nm and an emission wavelength of 535 nm.

Paracellular permeability was evaluated using the fluorescent marker, Lucifer yellow (LY, Lucifer yellow CH, lithium salt, Thermo Scientific). A transport buffer composed of 1%v/v HEPES solution added to Hank’s Balanced Salt Solution (HBSS) (1×, Gibco) was added to wells of a 24-well plate and placed in a humidified incubator at 37°C, 5% CO2. The hanging cell culture inserts were then rinsed with the transport buffer and then placed in the 24-well plate with the transport buffer. LY solution (300 μM) was then added to the apical side of the inserts (0.3 ml per insert) and the plate was incubated at 37°C and 5% CO₂ for 1.30 h. Samples taken from both the apical and basolateral sides of each insert were then analyzed using a Tecan Spark multimode microplate reader to record fluorescence values (excitation/emission wavelengths at 428/536 nm). A standard curve was then used to estimate the LY concentration of each sample and then apparent permeability (Papp, cm/s) was calculated using:
LY_A and LY_B are the concentrations of LY in the apical and basal compartments, respectively. Vol_A and Vol_B are the volumes in the apical and basal sides, respectively. T (seconds) is the time of incubation. C is the initial concentration of LY on the apical side (300 μM) and A is the area of the membrane.