Fitc dextran

At 23 hrs post final instillation and 1 hr prior to harvest, 0.2 mg 4kDa FITC-dextran (Sigma#46944) was administered o.p. to mice. BALF was collected as above. Blood was collected via cardiac puncture, and centrifuged for 12 min at 12,000 rpm at 4°C to separate serum from cellular component. FITC-dextran levels were assessed using a fluorescent plate reader (Beckman Coulter DTX 880 Multimode plate reader).

Monolayers of human 16HBE bronchial epithelial cells passage 17–20 (a gift from Dr. D. C. Gruenert, University of California San Francisco, CA) were grown on Transwell inserts (Corning; polyester inserts with 0.4 um pores and 0.33 cm² growth area) then exposed to low-dose poly I:C (0.05 or 0.5 μg/ml, InvivoGen Cat#tlrl-pic; Version#11C21-MM), a synthetic analog of viral double stranded RNA. Barrier function was measured with trans-epithelial electrical resistance (TEER) at 6, 24, and 48 hrs after polyI:C treatment using a voltometer (World Precision Instruments EVOM2). At 48 hrs after treatment, 4 kDa fluorescein isothiocyanate (FITC) dextran (Sigma, used at 10 μg/ml) was applied apically and accumulation of FITC-dextran into the basal chamber was quantified with a Beckman Coulter DTX 880 Multimode fluorescent plate reader 2 hrs later. To assess the effects of selected medications on cell permeability, monolayers were treated with 1–10 μM budesonide (Sigma), montelukast (Sigma), or formoterol (Sigma) 18 hrs prior to polyI:C challenge.

RAW264.7 cells were incubated with varying concentrations of H. aurantium body wall fatty acids for 24 h. Then, the cells were collected and incubated with 1 mg/ml of FITC-dextran (Sigma-Aldrich, USA) at 37°C for 1 h. The phagocytosis reaction was stopped using ice-cold PBS and then the cells were washed three times with cold PBS. Cellular uptake of FITC-dextran was analyzed using a CytoFLEX Flow Cytometer (Beckman Coulter, Inc., USA).