Flow cytometric phase-resolved discrimination of damaged/dead cells by propidium iodide uptake in macrophages having phagocytized fluorescent microspheres

John A. Steinkamp; Yolanda E. Valdez; Bruce E. Lehnert

doi:10.1117/12.426730

10 May 2001 Flow cytometric phase-resolved discrimination of damaged/dead cells by propidium iodide uptake in macrophages having phagocytized fluorescent microspheres

John A. Steinkamp, Yolanda E. Valdez, Bruce E. Lehnert

Author Affiliations +

Proceedings Volume 4252, Advances in Fluorescence Sensing Technology V; (2001) https://doi.org/10.1117/12.426730
Event: BiOS 2001 The International Symposium on Biomedical Optics, 2001, San Jose, CA, United States

Abstract

Instilled particle burdens of uniform green-yellow fluorescent microspheres phagocytized by rat alveolar (lung) macrophages and cell viability, as indexed by propidium iodide uptake (red fluorescence), were assessed using flow cytometry. Since the spectral emission from phagocytized microspheres partially overlapped the propidium iodide fluorescence and interfered with the conventional flow cytometric measurement of damaged/dead cells without subtractive compensation, this caused errors when estimating the percentage of non-viable, propidium iodide positive, phagocytic macrophages. The interference was eliminated by employing phase-sensitive detection in the red fluorescence measurement channel based on differences in lifetimes between the fluorescent microspheres and propidium iodide. In addition, intrinsic cellular autofluorescence, whose fluorescence lifetime is approximately the same as the phagocytized microspheres, also was eliminated in the measurement process. Since there was no detectable spectral interference of propidium iodide in the green fluorescence (particle phagocytosis) measurement channel, conventional fluorescence detection was employed.

Citation Download Citation

John A. Steinkamp, Yolanda E. Valdez, and Bruce E. Lehnert "Flow cytometric phase-resolved discrimination of damaged/dead cells by propidium iodide uptake in macrophages having phagocytized fluorescent microspheres", Proc. SPIE 4252, Advances in Fluorescence Sensing Technology V, (10 May 2001); https://doi.org/10.1117/12.426730

ACCESS THE FULL ARTICLE

INSTITUTIONAL
Select your institution to access the SPIE Digital Library.

SELECT YOUR INSTITUTION

PERSONAL
Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.

PERSONAL SIGN IN

No SPIE Account? Create one

PURCHASE THIS CONTENT

SUBSCRIBE TO DIGITAL LIBRARY

50 downloads per 1-year subscription

Members: $195

Non-members: $335 ADD TO CART

25 downloads per 1 - year subscription

Members: $145

Non-members: $250 ADD TO CART

PURCHASE SINGLE ARTICLE

Includes PDF, HTML & Video, when available