Rhodamine 123

The relative cellular accumulation of tracer dye Rhodamine123 (Sigma) was determined using flow cytometry as described before.^{69 (link)} Briefly, MCF-7/A02 cells were collected 48 h after being treated with BKM120 and then incubated with 0.3 μM Rhodamine123 in RPMI1640 medium with constant shaking for 90 min at 37 °C. After washing twice with ice-cold PBS, the cells were resuspended in 500 μl ice-cold PBS. Rhodamine123 accumulation in cells was determined using a flow cytometer (BD FACSCanto II, Becton Dickinson). The cells were excited at 488 nm, and emission was measured at 530 nm.

P-gp functionality was assessed using rhodamine 123 (Sigma), a substrate for P-gp, and efflux transporter inhibitors as follows. Human iPS-ECs, which were co-cultured with C6 cells or incubated in C6CM, were washed with PBS and pre-incubated at 37°C for 1 hr with or without inhibitors (5 μM cyclosporine A (Wako) or 10 μM MK571 (Sigma)), followed by addition of rhodamine 123 to the upper chamber. After 1 hr, the fluorescence activity of the medium in the lower chamber was quantified on a plate reader [24 (link)].

Caco-2 cells and their derivatives, which were cultured on the cell culture inserts, were rinsed with HBSS. The cells were preincubated for 30 min with HBSS in the presence or absence of 10 µM Cyclosporin A (Sigma-Aldrich) as the inhibitor. To measure apical to basolateral (A to B) permeability, the 1.5 mL of HBSS containing 10 μM Rhodamine123 (Sigma-Aldrich) was added to the apical chamber, and 3.0 mL of HBSS was also added to the basolateral chamber. To measure basolateral to apical (B to A) permeability, the 3.0 mL of HBSS containing 10 μM Rhodamine123 (Sigma-Aldrich) was added to the basolateral chamber, and 1.5 mL of HBSS was also added to the apical chamber. After 90 min incubation at 37 °C in the presence or absence of the inhibitor, the solution was collected from the basolateral (A to B) or apical (B to A) chamber. The Rhodamine123 fluorescent signal was measured with a fluorescence plate reader (TriStar LB 941, Berthold Technologies) using 428 nm excitation and 535 nm emission filters. Rhodamine123 concentrations were calculated using the standard curve generated by serial dilution of Rhodamine123.
The ER of rhodamine 123 was calculated according following equation. $$\text{ER}=Papp\left(\text{B}\text{to}\text{A}\right)/Papp\left(\text{A}\text{to}\text{B}\right)$$

Mitochondrial mediated apoptotic effect of BGT on A549 cells was assessed by Rhodamine 123 (Sigma, USA) staining method. The cells were washed and stained with Rhodamine 123 (10 μg/ml, Sigma) for 10 min and analysed in fluorescence spectrophotometer and microscopy.

MFI was analysed with flow cytometry (Rhodamine 123, Sigma-Aldrich, MO, USA): results are expressed as mean fluorescent intensity (MFI) of Rhodamine 123 of three independent experiments. Fluorescence was acquired with CyAn ADP analyzer and Summit software (Beckman Coulter, Brea, CA, USA), then analysed with FlowJo software (version 7.6, Treestar Inc., Ashland, OR, USA).

The P-gp functionality was evaluated using rhodamine 123 (Sigma-Aldrich), a substrate of the P-gp transporter [19 (link),29 (link)]. Both channels were pretreated with 5 µM cyclosporine A (CsA, P-gp inhibitor, Sigma-Aldrich) for 1 h. rhodamine 123 was added to the vascular channel and incubated in the presence or absence of cyclosporine A at a flow rate of 120 µL/h. [¹³C₁₂] Sucrose (100 µg/mL) was simultaneously added to monitor the barrier integrity. Effluents were collected from the apical and basal channels and quantified using fluorescence intensity. The fluorescence was measured at 485/530 nm to quantify the rhodamine 123 concentration via a SynergyMX2 ELISA plate reader (BioTek Instruments, Winooski, VT, USA). The BBB permeability of rhodamine 123 and sucrose was measured in the presence or absence of the inhibitor.