Rho123

Bufalin, Doxorubicin (DOX), Rho-123, verapamil and Lucifer yellow were obtained from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA). Minimum Essential Media (MEM), fetal bovine serum (FBS), non-essential amino acids (NEAA), Ham's F-12K (Kaighn’s) Medium (F12K), and Hank's balanced salt solution (HBSS), were obtained from Gibco BRL (Carlsbad, CA, USA). RPMI 1640 and phosphate-buffered saline (PBS) were from Hyclone (Thermo Scientific, Logan, UT). Annexin V-FITC Apoptosis Detection Kit was purchased from BD Biosciences (Beijing, China). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Kumamoto, Japan). A ABCB1 ATPase assay system was purchased from Promega (Madison, WI, USA). The primary antibodies for ABCB1 (#13342) and β-actin (#3700) were sourced from Cell Signaling Technology (Boston, USA). The secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

To assess P‐gp activity in the BBB model, the culture medium in the microfluidic devices was changed to DMEM (glucose‐free) or EAPM with or without 5 × 10⁻⁶m of the P‐gp inhibitor Verapamil (Verapamil hydrochloride, Sigma‐Aldrich, Buchs, Switzerland) under OGD or normal conditions. After 30 min of incubation, 5 × 10⁻⁶m of P‐gp substrate Rhodamine 123 (Rho 123, Sigma‐Aldrich, Buchs, Switzerland) in (glucose‐free) medium were injected into the microfluidic devices after medium exchange. The platform was incubated for 2.5 h under OGD or normal conditions, and the Rhodamine 123 was subsequently removed and replaced by normal medium. Rhodamine 123 uptake of the ECs was imaged using a Nikon spinning‐disk confocal microscope with a 40× water‐immersion objective. Image processing was carried out using the NIS‐Elements software package.

The mitochondrial membrane potential was measured by flow cytometry using Rho123 (Sigma) staining, as described previously (Wang et al., 2014 (link)). Briefly, the cells were incubated with Rho123 (0.5 μg/mL) at 37°C for 25 min and then were harvested and washed twice with PBS, and a total of 10,000 events per sample was measured by a flow cytometer. The fluorescent signal intensity was analyzed with BD CellQuest Pro software.

Effects of chemical exposure on MMP was assessed using rhodamine123 (rho123) (sigma Aldrich, R8004) using the protocol described previously (van der Stel et al. 2020 (link)). Cells were seeded two days before exposure. At the day of exposure, cells were first co-stained with 200 ng/µl Hoechst (Life technologies, H1399) and 1 µM rho123 for 75 min, followed by a co-staining of 0.2 µM rho123 and 100 nM propidium iodide (PI) (Sigma-Aldrich, P4170) and exposure to the desired concentration of test chemical. Over a period of 24 h, the signal intensity of Hoechst (408 nm), rho123 (448 nm), and PI (561 nm) were monitored every hour. The nuclei were identified based on the Hoechst images and formed the basis for the assessment of the cytoplasm (defined as those pixels within a maximal distance of 10 pixels around the nucleus). Finally, the intensity of the rho123 was assessed in the cytoplasm, and cell death estimates were based on the fraction of nuclei that showed at least 10% of the pixels also positive for PI-staining. Part of the MMP data were used for development of a dynamic mathematical model and integration with RNA-seq data elsewhere (Yang et al. 2021 (link), van der Stel et al. 2021 (link)).

DL0410 (purity > 99%) was synthesized by professor Lin Wang and Song Wu in the institute of Materia Medica, Chinese Academy of Medical Sciences. Rho123 and dimethylsulfoxide (DMSO) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Verapamil and phenacetin (internal standard, IS) was obtained from the National Institutes for Food and Drug Control (Beijing, China). The ATPase Assay Kit was the product of Pgp-GloTM Assay, from Promega (V3601) (Madison, WI, USA). Acetonitrile and methanol were LC-MS grade and obtained from J.T. Baker (Seattle, WA, USA). Formic acid was HPLC-grade, purchased from TEDIA (Fairfield, CA, USA). Ethyl acetate (analytical grade) was obtained from Beijing Chemical Reagent Co. (Beijing, China). Pure water was purchased from Wahaha Company (Hangzhou, China).

Saccharomyces cerevisiae YPD-grown cultures were harvested and suspended in PBS at 1×10⁷ cell/mL and loaded with the fluorescent probe DHE or PGFL as detailed above, incubating with light shaking in darkness. Suspensions were treated with and without ethanol (10%) and incubated for 30 min at 30°C. Afterwards, the cell suspensions were incubated with Rhodamine 123 (Rho123; Sigma) during 30 min for mitochondrial co-localization and analyzed using a confocal microscope (Olympus FV1000). The signal evaluating fluorescence emission was observed between 560–580 nm for DHE, between 405–505 nm for PGFL and between 590–600 nm for Rho123. Images were acquired with different magnifications.