A high‐throughput 3‐parameter flow cytometry‐based cell death assay

Buenz, Eric J.; Limburg, Paul J.; Howe, Charles L.

doi:10.1002/cyto.a.20376

Cited by 14 publications

(7 citation statements)

References 21 publications

(30 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Cell cycle analysis was also performed on the Jurkat cells using Hoechst 33342, as previously described (Buenz et al, 2007). This evaluation confirmed cell death, and it was possible to observe cells in G 0 /G 1 (Figure 2C; 1) and G 2 /M ( Figure 2C; 2) phases as well as to distinguish an early-stage apoptotic population ( Figure 2C; 3) and a late-stage apoptotic population ( Figure 2C; 4).…”

Section: Resultsmentioning

confidence: 99%

“…Previous work had established a highthroughput screen to evaluate the toxicity of botanical extracts or lead compounds using Jurkat T cells (Buenz et al, 2007). Jurkats are an accepted and commonly employed model for the examination of cell death (Buenz et al, 2006c;Buenz et al, 2004b), as they employ a well-defined apoptotic pathway (Buenz et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A high-throughput cell-based toxicity analysis of drug metabolites using flow cytometry

Buenz

2007

Cell Biol Toxicol

View full text Add to dashboard Cite

The effects of liver enzymes on drug activities are important considerations in the drug discovery process. Frequently, liver microsomes are used to simulate first-pass metabolism in the liver; however, there are significant disadvantages to the microsome system. As an alternative, a simple cell-based, high-throughput system that allows for examination of metabolite activity is described. Using multiparameter flow cytometry and the low-volume, high-sample format of 96-well plates, it is possible to rapidly evaluate a dose-response curve for metabolites based on variables including initial compound concentrations, hepatocyte cell line metabolic activities, and time. Using HepG2 cells as a surrogate for hepatic metabolism of a potential therapeutic, the impact of metabolites on Jurkat cell death was measured by both propidium iodide dye exclusion and cell cycle analysis. While this system is not proposed to supplant liver microsome studies, this alternative assay provides a highly adaptable, low-cost, and high-throughput measure of drug metabolism.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A high-throughput cell-based toxicity analysis of drug metabolites using flow cytometry

Buenz

2007

Cell Biol Toxicol

View full text Add to dashboard Cite

show abstract

“…We routinely use flow cytometry to determine both the quality of the brain infiltrating cell preparation, and to distinguish different populations of immune cells 2, 9 . At acute time-points, our BILs method yields high percentages of inflammatory monocytes within the CD45hi population as well as high percentages of macrophages in the CD45lo population.…”

Section: Discussionmentioning

confidence: 99%

Isolation of Brain-infiltrating Leukocytes

LaFrance‐Corey

Howe

2011

JoVE

Self Cite

View full text Add to dashboard Cite

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler’s murine encephalomyelitis virus (TMEV) via a rapid intracranial injection technique and how to purify a leukocyte-enriched population of infiltrating cells from whole brain. Briefly, mice are anesthetized with isoflurane in a closed chamber and are free-hand injected with a Hamilton syringe into the frontal cortex. Mice are then killed at various times after infection by isoflurane overdose and whole brains are extracted and homogenized in RPMI with a Tenbroeck tissue grinder. Brain lysates are centrifuged through a continuous 30% Percoll gradient to remove the myelin and other cell debris. The cell suspension is then strained at 40 μm, washed and centrifuged on a discontinuous Ficoll-Paque Plus gradient to select and purify the leukocytes. The leukocytes are then washed and resuspended in appropriate buffers for immunophenotyping by flow cytometry. Flow cytometry reveals a population of innate immune cells at the early stages of infection in C57BL/6 mice. At 24 hours post infection, multiple subsets of immune cells are present in the BILs, with an enriched population of Gr1+, CD11b+ and F4/80+cells. Therefore, this method is useful in characterizing the immune response to acute infection in the brain.

show abstract

“…Jurkat cells were incubated with D. seychellarum extract (10, 50, 100, 250, 500, 1000 μg/mL) for 18 hr in the tissue culture incubator as previously described (Buenz et al, 2007). To each sample, propidium iodide (PI;Sigma; St. Louis, MO) was added to achieve a final concentration of 10 ng/mL.…”

Section: Cell Death and Mitochondrial Membrane Potential (δψM) By Cytmentioning

confidence: 99%

Cytotoxic Properties of Diospyros Seychellarum Extract

Buenz

Bauer²,

Motley

et al. 2007

J. Toxicol. Sci.

Self Cite

View full text Add to dashboard Cite

-During a recent botanical expedition in the Seychelles archipelago we identified healers using Diospyros seychellarum as a tonic. Since this plant lacks any medicinal record in the current literature, we assessed the cytotoxic potential of D. seychellarum. Using Jurkat cells as a model system we show, by flow cytometry, that treatment with the leaf extract results in mitochondrial depolarization and subsequent loss of cellular membrane integrity. Additionally, by transmission electron microscopy, we show that treatment with the extract results in chromatin condensation, mitochondrial swelling, and loss of nuclear membrane integrity. Through these morphological and biochemical observations we concluded that the extract of Diospyros seychellarum is able to induce apoptosis. While it is difficult to extrapolate a potential pharmacologic function based on the ethnomedical use as a tonic, the ability of this extract to induce apoptosis warrants further investigation of the medicinal properties of this plant.

show abstract

A high‐throughput 3‐parameter flow cytometry‐based cell death assay

Cited by 14 publications

References 21 publications

A high-throughput cell-based toxicity analysis of drug metabolites using flow cytometry

A high-throughput cell-based toxicity analysis of drug metabolites using flow cytometry

Isolation of Brain-infiltrating Leukocytes

Cytotoxic Properties of Diospyros Seychellarum Extract

Contact Info

Product

Resources

About