Determination of quantitative parameters of Escherichia coli phagocytosis by mouse peritoneal macrophages

Miliukienė, Valė; Biziulevičienė, Gene; Chaustova, Larisa; Pilinkienë, A.; Biziulevičius, Gediminas A.

doi:10.1134/s1990519x07050112

Cited by 12 publications

(11 citation statements)

References 9 publications

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“…Similar to TB, PI has the ability to penetrate into dead cells and to complex with DNA ( 14 - 16 ). We propose a TB exclusion test using flow cytometry as an alternative, cheap, and reliable technique to be used together with phenotype analysis, with no interference with cell viability and no quenching of green fluorescence, as has been previously reported ( 17 ).…”

Section: Introductionmentioning

confidence: 79%

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Trypan blue exclusion assay by flow cytometry

Avelar-Freitas

Almeida

Pinto

et al. 2014

Braz J Med Biol Res

132

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Dye exclusion tests are used to determine the number of live and dead cells. These assays are based on the principle that intact plasma membranes in live cells exclude specific dyes, whereas dead cells do not. Although widely used, the trypan blue (TB) exclusion assay has limitations. The dye can be incorporated by live cells after a short exposure time, and personal reliability, related to the expertise of the analyst, can affect the results. We propose an alternative assay for evaluating cell viability that combines the TB exclusion test and the high sensitivity of the flow cytometry technique. Previous studies have demonstrated the ability of TB to emit fluorescence when complexed with proteins. According to our results, TB/bovine serum albumin and TB/cytoplasmic protein complexes emit fluorescence at 660 nm, which is detectable by flow cytometry using a 650-nm low-pass band filter. TB at 0.002% (w/v) was defined as the optimum concentration for distinguishing unstained living cells from fluorescent dead cells, and fluorescence emission was stable for 30 min after cell treatment. Although previous studies have shown that TB promotes green fluorescence quenching, TB at 0.002% did not interfere with green fluorescence in human live T-cells stained with anti-CD3/fluorescein isothiocyanate (FITC) monoclonal antibody. We observed a high correlation between the percentage of propidium iodide+CD3/FITC+ and TB+CD3/FITC+ cells, as well as similar double-stained cell profiles in flow cytometry dot-plot graphs. Taken together, the results indicate that a TB exclusion assay by flow cytometry can be employed as an alternative tool for quick and reliable cell viability analysis.

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Section: Introductionmentioning

confidence: 79%

“…To evaluate whether TB promotes quenching of the green fluorescence as previously reported ( 17 ), 100 µL heparinized blood samples was incubated with 2 µL undiluted FITC-conjugated monoclonal anti-human CD3 antibody (Cat. No.…”

Section: Methodsmentioning

confidence: 99%

Trypan blue exclusion assay by flow cytometry

Avelar-Freitas

Almeida

Pinto

et al. 2014

Braz J Med Biol Res

132

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“…Staining of macrophages for markers such as CD11b and F4/80 was carried out in FACS buffer, and we found that this had a negative effect on survival of bacteria. A similar result was seen where sodium azide caused inhibition of E.coli phagocytosis in murine peritoneal macrophages (Miliukiene et al, 2007). Therefore using FACS buffer would skew the phagocytic results.…”

Section: Discussionsupporting

confidence: 65%

The Effects of Microtubule Stabilizing Drugs on Macrophage Immune-Mediated Endocytosis

Joshi¹

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<p>Microtubule stabilizing drugs (MSD) bind and stabilize microtubules, thus inhibiting their normal function. MSD exhibit anti-mitotic effects which makes them attractive as cancer chemotherapeutics and much of existing research has focused on these effects in proliferating cells. In contrast, we are interested in assessing the effects of microtubule stabilization on non-proliferating cells, such as macrophages, to determine potential mitosis-independent actions of MSD on microtubule function. Thus, we investigated the effects of MSD on macrophage receptor-mediated endocytosis of low density lipoproteins (LDL) and found no significant effect on the ability of paclitaxel-treated macrophages to endocytose LDL. Alterations to macrophage phagocytic and killing efficiency due to treatment with paclitaxel, peloruside or docetaxel, as well as the recently discovered compounds, ixabepilone, mycothiazole, and zampanolide were investigated. Treatment with paclitaxel, peloruside or docetaxel did not significantly inhibit phagocytosis or killing of bacteria. Results from confocal microscopy suggest that paclitaxel alters phagocytic kinetics in macrophages. Respectively, zampanolide and mycothiazole significantly inhibited macrophage bactericidal and killing ability, while Ixabepilone enhanced bacterial killing. MSD treatment also altered production of tumor necrosis factor alpha (TNF-a) and nitric oxide (NO) during bacterial killing. Optimal activation of macrophages with IFN-y did not alter the effects of MSD. Taken together, these results suggest that MSD have multiple immunomodulatory effects unrelated to their anti-mitotic effects. The data suggests that during MSD treatment, macrophage activity maybe altered or impaired, thus modifying the ability of patients to fight off bacterial infections.</p>

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“…Macrophages were derived from the bone marrow (BM) ( Ondee et al, 2017 ) and phagocytosis assay was performed following an established protocol ( Chmiela et al, 1997 ; Miliukene et al, 2007 ). In short, 1 × 10 9 cell/ml heat-killed HP (60°C for 30 min) was incubated with 100 μg/ml fluorescein isothiocyanate (FITC) (Sigma, United States) in PBS at 35°C for 30 min for FITC-labeling.…”

Section: Methodsmentioning

confidence: 99%

Helicobacter pylori Infection Increased Anti-dsDNA and Enhanced Lupus Severity in Symptomatic FcγRIIb-Deficient Lupus Mice

et al. 2018

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The defect on Fc gamma receptor IIb (FcγRIIb), the only inhibitory FcγR, has been identified as one of the genetic factors increasing susceptibility to lupus. The prevalence of Helicobacter pylori (HP) and FcγRIIb dysfunction-polymorphisms are high among Asians, and their co-existence is possible. Unfortunately, the influence of HP against lupus progression in patients with lupus is still controversial. In this study, the interactions between these conditions were tested with HP infection in 24-week-old FcγRIIb-/- mice (symptomatic lupus). HP induced failure to thrive, increased stomach bacterial burdens and stomach injury (histology and cytokines) in both wild-type and FcγRIIb-/- mice. While the severity of HP infection, as determined by these parameters, was not different between both strains, antibodies production (anti-HP, anti-dsDNA and serum gammaglobulin) were higher in FcγRIIb-/- mice compared to wild-type. Accordingly, HP infection also accelerated the severity of lupus as determined by proteinuria, serum creatinine, serum cytokines, renal histology, and renal immune complex deposition. Although HP increased serum cytokines in both wild-type and FcγRIIb-/- mice, the levels were higher in FcγRIIb-/- mice. As such, HP also increased spleen weight and induced several splenic immune cells responsible for antibody productions (activated B cell, plasma cell and follicular helper T cell) in FcγRIIb-/- mice, but not in wild-type. These data describe the different systemic responses against localized HP infection from diverse host genetic background. In conclusion, the mutual interactions between HP and lupus manifestations of FcγRIIb-/-mice were demonstrated in this study. With the prominent immune responses from the loss of inhibitory signaling in FcγRIIb-/- mice, HP infection in these mice induced intense chronic inflammation, increased antibody production, and enhanced lupus severity. Thus, the increased systemic inflammatory responses due to localized HP inducing gastritis in some patients with lupus may enhance lupus progression. More studies are needed.

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Determination of quantitative parameters of Escherichia coli phagocytosis by mouse peritoneal macrophages

Cited by 12 publications

References 9 publications

Trypan blue exclusion assay by flow cytometry

Trypan blue exclusion assay by flow cytometry

The Effects of Microtubule Stabilizing Drugs on Macrophage Immune-Mediated Endocytosis

Helicobacter pylori Infection Increased Anti-dsDNA and Enhanced Lupus Severity in Symptomatic FcγRIIb-Deficient Lupus Mice

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