F-actin Imaging of Mechanically Compressed Pseudostratified Human Bronchial Epithelial Cells

Overview

This dataset includes immunofluorescence microscopy of pseudostratified airway epithelial cells stained for F-actin. Each field of view consists of 3 images visualizing the apical cell boundaries, basal cell boundaries, and basal cell stress fibers.

Cell Culture and Treatment

Primary human bronchial epithelial cells (from a single donor) were grown on transwells in air-liquid interface culture for 14 days to model a well-differentiated, pseudostratified airway epithelium. Cells were then exposed to mechanical compression (30 cmH2O for 3 hours) mimicking asthmatic bronchoconstriction. Cells were fixed (4% PFA for 30 minutes) at 24, 48, or 72 hours after mechanical compression. Two transwells were collected per condition and timepoint.

Immunofluorescence Imaging

Fixed transwells were stained for F-actin (Alexa fluor 488-Phalloidin, ThermoFisher Scientific, diluted 1:40, 30 minutes). Transwell membranes were cut from the plastic support and mounted on glass slides. Slides were imaged using a Zeiss Axio Observer Z1 with an apotome module controlled using Zen Blue 2.0 software. Five random fields of view were imaged from each transwell membrane in a z-stack from substrate to apical cell surface. To visualize various planes through the pseudostratified epithelial layer (apical cell boundaries, basal cell boundaries, and basal cell stress fibers), maximum intensity projections were generated from regions of interest through the z-stack.

Dataset

Each image file contains

Timepoint: 24, 48, or 72 hours after treatment

Condition & Well: control (C) or mechanical compression (P) followed by a number indicating the well (1 or 2)

Unique Z-stack ID: 3-digit number

Miscellaneous note: MIP or MIP_ROI

Region of Interest: apical cell boundaries (ACB), basal cell boundaries (BCB), or basal stress fibers (SF)

For example, these 3 maximum intensity projection images came from the same z-stack/field of view. They are from the 72 hour timepoint, mechanical compression, well #2, z-stack #147:

72hr_P2_147_MIP_ACB.tif: apical cell boundaries

72hr_P2_147_MIP_ROI_BCB.tif: basal cell boundaries

72hr_P2_147_MIP_ROI_SF.tif: basal stress fiber