Tools to Measure Cell Health and Cytotoxicity Using High Content Imaging and Analysis

Mandavilli, Bhaskar S.; Aggeler, Robert; Chambers, Kevin M.

doi:10.1007/978-1-4939-7357-6_3

Cited by 9 publications

(4 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Endpoints and the multiplex fluorescent staining used are described in Table 1 and below. Assays were conducted based on modifications to previously described standardized methods or strictly following fluorescent dye manufacturers’ protocols [ 48 , 49 ]. If recommended by dye manufacturer or to reduce high background signal, stained cells were washed with 37 °C fresh culture medium then imaged in clear Fluorobrite DMEM (ThermoFisher).…”

Section: Methodsmentioning

confidence: 99%

In vitro inflammation and toxicity assessment of pre- and post-incinerated organomodified nanoclays to macrophages using high-throughput screening approaches

Stueckle,

Jensen,

Coyle

et al. 2024

Part Fibre Toxicol

View full text Add to dashboard Cite

Background Organomodified nanoclays (ONC), two-dimensional montmorillonite with organic coatings, are increasingly used to improve nanocomposite properties. However, little is known about pulmonary health risks along the nanoclay life cycle even with increased evidence of airborne particulate exposures in occupational environments. Recently, oropharyngeal aspiration exposure to pre- and post-incinerated ONC in mice caused low grade, persistent lung inflammation with a pro-fibrotic signaling response with unknown mode(s) of action. We hypothesized that the organic coating presence and incineration status of nanoclays determine the inflammatory cytokine secretary profile and cytotoxic response of macrophages. To test this hypothesis differentiated human macrophages (THP-1) were acutely exposed (0–20 µg/cm2) to pristine, uncoated nanoclay (CloisNa), an ONC (Clois30B), their incinerated byproducts (I-CloisNa and I-Clois30B), and crystalline silica (CS) followed by cytotoxicity and inflammatory endpoints. Macrophages were co-exposed to lipopolysaccharide (LPS) or LPS-free medium to assess the role of priming the NF-κB pathway in macrophage response to nanoclay treatment. Data were compared to inflammatory responses in male C57Bl/6J mice following 30 and 300 µg/mouse aspiration exposure to the same particles. Results In LPS-free media, CloisNa exposure caused mitochondrial depolarization while Clois30B exposure caused reduced macrophage viability, greater cytotoxicity, and significant damage-associated molecular patterns (IL-1α and ATP) release compared to CloisNa and unexposed controls. LPS priming with low CloisNa doses caused elevated cathepsin B/Caspage-1/IL-1β release while higher doses resulted in apoptosis. Clois30B exposure caused dose-dependent THP-1 cell pyroptosis evidenced by Cathepsin B and IL-1β release and Gasdermin D cleavage. Incineration ablated the cytotoxic and inflammatory effects of Clois30B while I-CloisNa still retained some mild inflammatory potential. Comparative analyses suggested that in vitro macrophage cell viability, inflammasome endpoints, and pro-inflammatory cytokine profiles significantly correlated to mouse bronchioalveolar lavage inflammation metrics including inflammatory cell recruitment. Conclusions Presence of organic coating and incineration status influenced inflammatory and cytotoxic responses following exposure to human macrophages. Clois30B, with a quaternary ammonium tallow coating, induced a robust cell membrane damage and pyroptosis effect which was eliminated after incineration. Conversely, incinerated nanoclay exposure primarily caused elevated inflammatory cytokine release from THP-1 cells. Collectively, pre-incinerated nanoclay displayed interaction with macrophage membrane components (molecular initiating event), increased pro-inflammatory mediators, and increased inflammatory cell recruitment (two key events) in the lung fibrosis adverse outcome pathway.

show abstract

Section: Methodsmentioning

confidence: 99%

In vitro inflammation and toxicity assessment of pre- and post-incinerated organomodified nanoclays to macrophages using high-throughput screening approaches

Stueckle,

Jensen,

Coyle

et al. 2024

Part Fibre Toxicol

View full text Add to dashboard Cite

show abstract

“…Research with bipolar patients and animal models observed a reduction in TCA cycle enzymes (Lee et al, 2007;Valvassori et al, 2013). In oxidative stress, it is essential to note that mitochondria are the primary source of free radicals, and are generated mainly through the ETC, during the energy production from glucose and oxygen, it generates oxidative stress (Barbosa et al, 2010;Mandavilli et al, 2018). Initially, we will describe the main processes, functions, and losses in oxidative stress, and later, the changes that occur in BD, which are related to the section on biomarkers.…”

Section: Energy Deficits In Neuronal Mitochondria and Its Possible Rementioning

confidence: 99%

Concepts of Neuroinflammation and Their Relationship With Impaired Mitochondrial Functions in Bipolar Disorder

Cyrino

Lima

Ullmann

et al. 2021

Front. Behav. Neurosci.

View full text Add to dashboard Cite

Bipolar disorder (BD) is a chronic psychiatric disease, characterized by frequent behavioral episodes of depression and mania, and neurologically by dysregulated neurotransmission, neuroplasticity, growth factor signaling, and metabolism, as well as oxidative stress, and neuronal apoptosis, contributing to chronic neuroinflammation. These abnormalities result from complex interactions between multiple susceptibility genes and environmental factors such as stress. The neurocellular abnormalities of BD can result in gross morphological changes, such as reduced prefrontal and hippocampal volume, and circuit reorganization resulting in cognitive and emotional deficits. The term “neuroprogression” is used to denote the progressive changes from early to late stages, as BD severity and loss of treatment response correlate with the number of past episodes. In addition to circuit and cellular abnormalities, BD is associated with dysfunctional mitochondria, leading to severe metabolic disruption in high energy-demanding neurons and glia. Indeed, mitochondrial dysfunction involving electron transport chain (ETC) disruption is considered the primary cause of chronic oxidative stress in BD. The ensuing damage to membrane lipids, proteins, and DNA further perpetuates oxidative stress and neuroinflammation, creating a perpetuating pathogenic cycle. A deeper understanding of BD pathophysiology and identification of associated biomarkers of neuroinflammation are needed to facilitate early diagnosis and treatment of this debilitating disorder.

show abstract

“…Notably, hypoxic areas in tumours often lead to lower efficiency of radio-therapies [6]. The investigation of hypoxia is challenging to establish due to the low availability of sensitive fluorescent dyes [7], explicitly measuring the activity of hypoxic cells.…”

Section: Introductionmentioning

confidence: 99%

Focused Ultrasound Treatment of a Spheroid In Vitro Tumour Model

Landgraf

Kozlowski

Zhang

et al. 2022

Cells

View full text Add to dashboard Cite

Focused ultrasound (FUS) is a non-invasive technique producing a variety of biological effects by either thermal or mechanical mechanisms of ultrasound interaction with the targeted tissue. FUS could bring benefits, e.g., tumour sensitisation, immune stimulation, and targeted drug delivery, but investigation of FUS effects at the cellular level is still missing. New techniques are commonly tested in vitro on two-dimensional (2D) monolayer cancer cell culture models. The 3D tumour model—spheroid—is mainly utilised to mimic solid tumours from an architectural standpoint. It is a promising method to simulate the characteristics of tumours in vitro and their various responses to therapeutic alternatives. This study aimed to evaluate the effects of FUS on human prostate and glioblastoma cancer tumour spheroids in vitro. The experimental follow-up enclosed the measurements of spheroid integrity and growth kinetics, DNA damage, and cellular metabolic activity by measuring intracellular ATP content in the spheroids. Our results showed that pulsed FUS treatment induced molecular effects in 3D tumour models. With the disruption of the spheroid integrity, we observed an increase in DNA double-strand breaks, leading to damage in the cancer cells depending on the cancer cell type.

show abstract

Tools to Measure Cell Health and Cytotoxicity Using High Content Imaging and Analysis

Cited by 9 publications

References 36 publications

In vitro inflammation and toxicity assessment of pre- and post-incinerated organomodified nanoclays to macrophages using high-throughput screening approaches

In vitro inflammation and toxicity assessment of pre- and post-incinerated organomodified nanoclays to macrophages using high-throughput screening approaches

Concepts of Neuroinflammation and Their Relationship With Impaired Mitochondrial Functions in Bipolar Disorder

Focused Ultrasound Treatment of a Spheroid In Vitro Tumour Model

Contact Info

Product

Resources

About