Because scientific study Tat binds strongly to serum proteins, all experiments were carried out in serum free media. D407 cells remained healthy and viable under these experimental conditions. The Tat treatment in the present study involved exposing D407 cells exposure to 100 nM Tat for 24 hours, which has frequently been used in previous in vitro studies. Cell viability assay Cells were grown in 96 well plates at a density of 1 104 cells well. After the indicated treatments, MTT was added at 5 mg ml to each well for 4 hours, after which the culture medium was removed and 150 l of DMSO was added to each well. The absorbance was measured at 490 nm using a multifunctional microplate reader. Measurement of TER Transparent Millicell CM filters were coated with 50 l of a rat tail collagen I ethanol mixture and left to dry before cells were subcul tured.

D407 cells were seeded at a density of 104 cells filter on the filters was supported by 24 well culture plates. The volumes on the apical and basolateral side were 400 l and 600 l, respec tively. The fluid pressure was the same in the two cham bers. The cultures were incubated in a humidified atmosphere. The medium was changed on the fol lowing day, and subsequently changed every second day for the duration of the experiment. Phase contrast micro scopy revealed that cells reached confluence at day 3, and then serum concentration of the culture medium was reduced to 1%. From 2 days after seeding, the TER was measured by an epithelial voltohmeter every other day to monitor the time course of the TER.

We began the indicated treat ments at day 10, the culture medium in control group also changed into serum free, and measured the TER at 1, 2, 3, 12, 24, 48, and 72 hours after exposure to 100 nM Tat. Permeability assay The paracellular permeability of RPE cells was determined by measuring the apical to basolateral movement of sodium fluorescein, using a slightly modi fied version of the technique of Hartnett et al. Briefly, to assess the fluid flux across the monolayer, sodium flu orescein mixed in DMEM was added to the apical compartment of the inserts after the indicated treatment. 100 l of fluid was collected from the basolateral compartment of each filter at 20, 40 and 60 min after adding sodium fluorescein, and then trans ported to 96 well black culture plates to measure the fluorescence.

The same volume of the appropriate medium was added to replace the medium removed. The fluorescence was measured by a multifunctional Dacomitinib microplate reader. The basolateral to total fluorescence ratio was determined for each group, and expressed as a percentage, with larger percentage indicating greater per meability. The fluorescence of DMEM mixed with 25 mg ml sodium fluorescein was taken as the total fluorescence. Real time reverse transcriptase polymerase chain reaction Total RNA was isolated with TRIzol reagent.