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

Md Ibrahim; Sakib M Moinuddin; Md Shahadat Hossain; Tri Nguyen; Tanoy Sarkar; Ahmed El-Shamy; Luca Cucullo; Tim Lahm; Eva Nozik; Marc A Simon; Kurt R Stenmark; Fakhrul Ahsan

doi:10.1016/j.xpro.2025.104147

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

STAR Protoc. 2025 Oct 21;6(4):104147. doi: 10.1016/j.xpro.2025.104147. Online ahead of print.

Authors

Affiliations

¹ Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, California Northstate University, Elk Grove, CA 95757, USA; Masters of Pharmaceutical Sciences Program, College of Graduate Studies, California Northstate University, Elk Grove, CA 95757, USA; Oncovask Therapeutics LLC, Elk Grove, CA 95758, USA. Electronic address: md.ibrahim@cnsu.edu.
² Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, California Northstate University, Elk Grove, CA 95757, USA.
³ Masters of Pharmaceutical Sciences Program, College of Graduate Studies, California Northstate University, Elk Grove, CA 95757, USA.
⁴ Oakland University William Beaumont School of Medicine, 586 Pioneer Dr, Rochester, MI 48309, USA.
⁵ Division of Pulmonary, Critical Care and Sleep Medicine, National Jewish Health, Denver, CO 80206, USA.
⁶ Department of Pediatrics, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA.
⁷ Division of Cardiology, Department of Medicine University of California, San Francisco, San Francisco, CA 94143, USA.
⁸ Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, California Northstate University, Elk Grove, CA 95757, USA; Veterans Affairs Northern California Health Care System, Martinez, CA 94553, USA. Electronic address: fakhrul.ahsan@cnsu.edu.

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.

Keywords: Biotechnology and bioengineering; Cell Biology; Cell culture; High Throughput Screening; Microscopy; Molecular Biology.