Phase Contrast Time-Lapse and F-actin Imaging of Mechanically Compressed or Irradiated Pseudostratified Human Bronchial Epithelial Cells

Overview

This dataset includes phase contrast time-lapse imaging of in vitro pseudostratified airway epithelial cells to visualize their collective cellular migration after exposure to mechanical compression (mimicking bronchoconstriction) or irradiation. Additionally, the cells were fixed and stained for F-actin to visualize 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 (ALI) culture for 14 days to model a well-differentiated, pseudostratified airway epithelium. Cells were then exposed to either mechanical compression (30 cmH2O for 3 hours) mimicking asthmatic bronchoconstriction or irradiation (1Gy of ionizing radiation using a RS 2000 Biological Research Irradiator (RadSource) on ALI days 7, 10, and 14).

Phase Contrast Time-Lapse Imaging

At 24 or 72 hours after final treatment, cells were imaged to visualize collective cellular migration. For each independent experimental replicate (2 transwells per treatment per timepoint), six fields of view per well were imaged every 6 minutes over 1.5 hours. The imaging chamber was supplied with 37°C, 5% CO2, humidified air on a Zeiss Axio Observer Z1 to collect phase contrast images. The image resolution is 0.586 µm/pixel.

Immunofluorescence Imaging

Cells were fixed (4% PFA for 30 minutes) at 24 or 72 hours after final treatment (and after phase contrast time-lapse 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. The image resolution is 0.293 µm/pixel.

Dataset

Phase contrast time-lapse movies are provided as *.avi files. Immunofluorescence images are provided as *.tif files. For an individual transwell, the imaging dataset includes:

6 phase contrast time-lapse movies

5 immunofluorescence images of apical cell boundaries

5 immunofluorescence images of basal cell boundaries

5 immunofluorescence images of basal cell stress fibers

Phase contrast time-lapse filenames contain

Donor: U13

Timepoint: 24 or 72 hours

Treatment & Well: control (C), mechanical compression (P), or irradiation (R); well 1 or 2

Field of View: (1) – (6)

Immunofluorescence image filenames contain:

Donor: U13

Timepoint: 24 or 72 hours

Treatment & Well: control (C), mechanical compression (P), or irradiation (R); well 1 or 2

Field of View: 1-5

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

Phase contrast time-lapse and immunofluorescence from the same transwell will all start with the same “Donor_Timepoint_Treatment/Well...” (i.e. U13_24_C1…). Note that the images from phase contrast and immunofluorescence are not necessarily from matched locations within the transwell and are at different spatial scales.

Immunofluorescence images from the same z-stack field of view will start with the same “Donor_Timepoint_Treatment/Well_FieldofView…” (i.e. U13_24_C1_1…).