Supplementary Information s37

Supplementary Information

Functional and cytometric examination of different human lung epithelial cell types as drug transport barriers

Kyoung Ah Min1,2 · Gus R. Rosania2 · Chong-Kook Kim3 · Meong Cheol Shin1,*

1 College of Pharmacy and Research Institute of Pharmaceutical Sciences, Gyeongsang National University, 501 Jinju Daero, Jinju, Gyeongnam 52828, Republic of Korea

2 Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, 428 Church St., Ann Arbor, MI 48109, USA

3 College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea

Supplementary data

C Users Admin Documents image analysis BBRC 2015 Supplementary data Figure S3 1 tif

Fig. S1. Confocal fluorescence microscopic images of three different human lung epithelial cells ((A) NHBE, (B) NL-20, (C) Calu-3) after dye transport in different days of cultures (day 3, 6, 8, 10, and day 20) under ALI condition. The cells on the Transwell™ porous membrane (pore size: 3 μm) were incubated with the mixture of cell-permeant probes (1 μM MTR (red), 10 μg/mL Hoe (blue), 2.5 μM LTG (green)) in the apical side and then underwent confocal analyses with Zeiss LSM 510 confocal microscopy. Three axis, x, y, and z were depicted with the arrows in the images processed by Zeiss LSM browser. Cell architectures in the inserts are apparently shown in xz planes with the indications of the arrows for the top, bottom cell layer and the porous membrane. Scale bar = 20 µm.

Fig. S2. Relationship of quantitative 2D and 3D parameters in normal human lung epithelial cells, NHBE. From the confocal images of NHBE cells, morphometric parameters such as cell area (μm2), height (μm), and volume (μm3) were measured by MetaMorph software for the cells on (A) top or (B) bottom layers in Transwell™ inserts. Coupling of cell area vs. height, cell area vs. volume, or cell height vs. volume were shown in the plots.

Fig. S3. Relationship of quantitative 2D and 3D parameters in NL-20 and Calu-3 cells. From the confocal cell images, morphometric parameters such as cell area (μm2), height (μm), and volume (μm3) were measured by MetaMorph software for the NL-20 cells on (A) top or (B) bottom layers and (C) Calu-3 cells in Transwell™ inserts. The scatter plots show the coupling of the cytometric parameters, cell area vs. height, cell area vs. volume, or cell height vs. volume.