Protocols for Studying Ocular Cell Volume Changes in Aqueous Humor Outflow Regulating Cells In Vitro
- By Dorette Z. Ellis1
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View Affiliations Hide Affiliations1 Department of Pharmaceutical Sciences, University of North Texas System College of Pharmacy, North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Worth, Texas, USA
- Source: Research Protocols for Ophthalmic Disease Mechanisms and Therapeutics: Glaucoma - Ocular Hypertension , pp 195-207
- Publication Date: August 2025
- Language: English
Protocols for Studying Ocular Cell Volume Changes in Aqueous Humor Outflow Regulating Cells In Vitro, Page 1 of 1
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This chapter describes a protocol for measuring trabecular meshwork and Schlemm's cell volume with different experimental conditions over time, utilizing fluorescence and confocal microscopic methods. A fluorescence probe is applied to the cells, and a confocal microscope captures pixeled, z-stacked images with 8 bits of resolution. The images are converted from 8-bit to 1-bit or binary. Threshold values are determined using fluorescent latex beads of known diameter, and cell volume is analyzed. ImageJ software is used to calculate the number of voxels in the region of interest in the image stack. This methodology is of interest, as the outflow of aqueous humor is actively controlled by cellular and molecular mechanisms in the aqueous humor outflow pathway that includes the trabecular meshwork and Schlemm's canal. Several cellular mechanisms have been characterized by which changes in aqueous humor outflow can occur, including changes in the trabecular meshwork and Schlemm's canal cell volume.
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