# Image cytometry-based quantification protocol of human pulmonary arterial endothelial cells in lab-fabricated multichannel microfluidic devices

## TL;DR

This paper introduces a protocol for automated counting and viability analysis of pulmonary arterial cells using image cytometry in microfluidic devices.

## Contribution

The protocol standardizes PAC quantification in microfluidic systems using automated imaging and staining techniques.

## Key findings

- PACs can be effectively cultured and analyzed on microfluidic chips integrated into 6-well plates.
- Hoechst 33342 and propidium iodide staining enables reliable live/dead cell analysis.
- Automated imaging and software-assisted quantification streamline pulmonary vascular research workflows.

## Abstract

Here, we present a protocol for automated quantification and viability analysis of pulmonary arterial cells (PACs) using a cellular image cytometer. We describe steps for obtaining PACs from the Pulmonary Hypertension Breakthrough Initiative, culturing and seeding them on microfluidic chips, and integrating chips into 6-well plates. Cells are stained with Hoechst 33342 and propidium iodide for live/dead analysis. Automated imaging and software-assisted quantification standardize workflows and broaden applications in pulmonary vascular research.

For complete details on the use and execution of this protocol, please refer to Al Hilal et al.1

•Steps for automated imaging and counting of cells on microfluidic chips•Instructions for staining cells with Hoechst and propidium iodide dyes•Procedures for positioning microfluidic devices in standard 6-well plate formats•Guidance for setting up imaging protocols and software-assisted quantification

Steps for automated imaging and counting of cells on microfluidic chips

Instructions for staining cells with Hoechst and propidium iodide dyes

Procedures for positioning microfluidic devices in standard 6-well plate formats

Guidance for setting up imaging protocols and software-assisted quantification

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Here, we present a protocol for automated quantification and viability analysis of pulmonary arterial cells (PACs) using a cellular image cytometer. We describe steps for obtaining PACs from the Pulmonary Hypertension Breakthrough Initiative, culturing and seeding them on microfluidic chips, and integrating chips into 6-well plates. Cells are stained with Hoechst 33342 and propidium iodide for live/dead analysis. Automated imaging and software-assisted quantification standardize workflows and broaden applications in pulmonary vascular research.