December 1989
Volume 30, Issue 12
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Articles  |   December 1989
Kinetics of phagocytosis in trabecular meshwork cells. Flow cytometry and morphometry.
Author Affiliations
  • S Shirato
    Department of Ophthalmology, University of California Medical Center, San Francisco 94143.
  • C G Murphy
    Department of Ophthalmology, University of California Medical Center, San Francisco 94143.
  • E Bloom
    Department of Ophthalmology, University of California Medical Center, San Francisco 94143.
  • L Franse-Carman
    Department of Ophthalmology, University of California Medical Center, San Francisco 94143.
  • M T Maglio
    Department of Ophthalmology, University of California Medical Center, San Francisco 94143.
  • J R Polansky
    Department of Ophthalmology, University of California Medical Center, San Francisco 94143.
  • J A Alvarado
    Department of Ophthalmology, University of California Medical Center, San Francisco 94143.
Investigative Ophthalmology & Visual Science December 1989, Vol.30, 2499-2511. doi:
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      S Shirato, C G Murphy, E Bloom, L Franse-Carman, M T Maglio, J R Polansky, J A Alvarado; Kinetics of phagocytosis in trabecular meshwork cells. Flow cytometry and morphometry.. Invest. Ophthalmol. Vis. Sci. 1989;30(12):2499-2511.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Confluent human trabecular meshwork (HTM) cells from three different donors and at various stages of serial passage were fed fluorescein-labeled polystyrene beads. Phagocytosis was monitored for up to 6 days using flow cytometry, fluorescence microscopy, and morphometric calculations from comprehensive electron microscopic observations at key time points. During the first 4 hr after initiation of phagocytosis, the confluent endothelial monolayer lost its cohesiveness and became segregated into separate cells. During the first 3 days the cells underwent marked and progressive changes in shape and size. After 4 days, some cells detached from the dish, as necrotic debris and degenerative changes appeared. The kinetics of phagocytosis in this stable, confluent monolayer showed that recruitment (the percentage of cells which had ingested at least one bead) proceeded semilogarithmically, with 50% of the cells recruited by 8 hr and 97% by 96 hr. The time course of phagocytosis (ie, the average number of beads phagocytosed per cell) is described by a sigmoidlike curve, reaching half-maximum at 40 hr and maximum (about 500 beads per cell) at 96 hr. The rate of uptake (ie, the first derivative of the average number of beads per cell) reached a peak (nine beads per cell per hr) at 24 hr and then decelerated slowly over the next 5 days. Cytochalasin B treatment, as a control, reduced phagocytosis by approximately 70%. Flow cytometry, when combined with electron microscopy, should provide a useful tool to examine phagocytosis in HTM cells exposed to steroids and other hormones and drugs.

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