A phagocytosis assay for oxidized low-density lipoprotein versus immunoglobulin G-coated microbeads in human U937 macrophages

Vance, David T.; Dufresne, Jaimie; Florentinus-Mefailoski, Angelique; Tucholska, Monika; Trimble, William S.; Grinstein, Sergio; Marshall, John

doi:10.1016/j.ab.2016.01.007

Cited by 9 publications

(6 citation statements)

References 60 publications

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“…Although conflicting data exist with regard to the mode of modified LDL uptake, there is a consensus on a crucial step needed for this process to occur: actin cytoskeletal reorganization is required for oxLDL internalization. Pharmacological inhibition of F-actin formation using latrunculin B or cytochalasin D had been shown to cause a marked (80%) reduction in oxLDL internalization [52,93]. Additionally, activation of Rac1 and Cdc42 that leads to actin reorganization promoted uptake of CD36 into vesicles that were smaller than conventional induced macropinosomes [42].…”

Section: Macropinocytosis Modified Lipoprotein Uptake and Atherosclementioning

confidence: 99%

Constitutive and stimulated macropinocytosis in macrophages: roles in immunity and in the pathogenesis of atherosclerosis

Doodnauth

Grinstein

Maxson

2018

Phil. Trans. R. Soc. B

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Section: Macropinocytosis Modified Lipoprotein Uptake and Atherosclementioning

confidence: 99%

Constitutive and stimulated macropinocytosis in macrophages: roles in immunity and in the pathogenesis of atherosclerosis

Doodnauth

Grinstein

Maxson

2018

Phil. Trans. R. Soc. B

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“…When evaluating the effects of a treatment, the combination of the three measurements allows for the determination of treatment effects on phagocytosis and of treatment caused cytotoxicity. We tested cytochalasin D in the assay, for its known effect of blocking actin filament formation (Casella et al., 1981), thus inhibiting phagocytosis and other actin‐mediated cell activities (Meena et al., 2021; Melzer et al., 2019; Murai et al., 1993; Tilney & Portnoy, 1989; Vance et al., 2016). We observed that 24‐hr cytochalasin D treatment (Fig.…”

Section: Commentarymentioning

confidence: 99%

Microglial Phagocytosis/Cell Health High‐Content Assay

2023

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We report a microglial phagocytosis/cell health high-content assay that has been used to test small molecule chemical probes and support our drug discovery projects targeting microglia for Alzheimer's disease therapy. The assay measures phagocytosis and cell health (cell count and nuclear intensity) simultaneously in 384-well plates processed with an automatic liquid handler. The mix-and-read live cell imaging assay is highly reproducible with capacity to meet drug discovery research needs. Assay procedures take 4 days including plating cells, treating cells, adding pHrodo-myelin/membrane debris to cells for phagocytosis, staining cell nuclei before performing high-content imaging, and analysis. Three selected parameters are measured from cells: 1) mean total fluorescence intensity per cell of pHrodo-myelin/membrane debris in phagocytosis vesicles to quantify phagocytosis; 2) cell counts per well (measuring compound effects on proliferation and cell death); and 3) average nuclear intensity (measuring compound induced apoptosis). The assay has been used on HMC3 cells (an immortalized human microglial cell line), BV2 cells (an immortalized mouse microglial cell line), and primary microglia isolated from mouse brains. Simultaneous measurements of phagocytosis and cell health allow for the distinction of compound effects on regulation of phagocytosis from cellular stress/toxicity related changes, a distinguishing feature of the assay. The combination of cell counts and nuclear intensity as indicators of cell health is also an effective way to measure cell stress and compound cytotoxicity, which may have broad applications as simultaneous profiling measurements for other phenotypic assays.

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“…For instance, given that the cell lines used U937 and HeLa cells are in vitro models of cancer [ 16 , 17 , 46 ], possible regulation of NFAT5 by SIRT6, may translate into discovery of other novel regulatory steps leading to cancer. Moreover, since U937 monocytes were widely used as a model of ox-LDL loaded foam cells of atherosclerotic lesions [ 47 – 49 ], modulation of AR expression by SIRT6 could be used as an experimental method for modulation of atherogenesis, as well. Hence, the findings exhibited through the cell lines utilized, may in turn have possible medical outcomes in future.…”

Section: Discussionmentioning

confidence: 99%

SIRT6 Is a Positive Regulator of Aldose Reductase Expression in U937 and HeLa cells under Osmotic Stress: In Vitro and In Silico Insights

Timuçin

Başağa

2016

PLoS ONE

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SIRT6 is a protein deacetylase, involved in various intracellular processes including suppression of glycolysis and DNA repair. Aldose Reductase (AR), first enzyme of polyol pathway, was proposed to be indirectly associated to these SIRT6 linked processes. Despite these associations, presence of SIRT6 based regulation of AR still remains ambiguous. Thus, regulation of AR expression by SIRT6 was investigated under hyperosmotic stress. A unique model of osmotic stress in U937 cells was used to demonstrate the presence of a potential link between SIRT6 and AR expression. By overexpressing SIRT6 in HeLa cells under hyperosmotic stress, its role on upregulation of AR was revealed. In parallel, increased SIRT6 activity was shown to upregulate AR in U937 cells under hyperosmotic milieu by using pharmacological modulators. Since these modulators also target SIRT1, binding of the inhibitor, Ex-527, specifically to SIRT6 was analyzed in silico. Computational observations indicated that Ex-527 may also target SIRT6 active site residues under high salt concentration, thus, validating in vitro findings. Based on these evidences, a novel regulatory step by SIRT6, modifying AR expression under hyperosmotic stress was presented and its possible interactions with intracellular machinery was discussed.

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A phagocytosis assay for oxidized low-density lipoprotein versus immunoglobulin G-coated microbeads in human U937 macrophages

Cited by 9 publications

References 60 publications

Constitutive and stimulated macropinocytosis in macrophages: roles in immunity and in the pathogenesis of atherosclerosis

Constitutive and stimulated macropinocytosis in macrophages: roles in immunity and in the pathogenesis of atherosclerosis

Microglial Phagocytosis/Cell Health High‐Content Assay

SIRT6 Is a Positive Regulator of Aldose Reductase Expression in U937 and HeLa cells under Osmotic Stress: In Vitro and In Silico Insights

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