A Simple High-Content Cell Cycle Assay Reveals Frequent Discrepancies between Cell Number and ATP and MTS Proliferation Assays

Chan, Grace K.Y.; Kleinheinz, Tracy; Peterson, David; Moffat, John G.

doi:10.1371/journal.pone.0063583

Cited by 145 publications

(132 citation statements)

References 43 publications

Supporting

Mentioning

126

Contrasting

Order By: Relevance

“…For direct cell count-based cytotoxicity assay, cells were incubated with ADC for 3 days (unless indicated otherwise) and then fixed, permeabilized, and nuclei-stained in one step, as described in ref. 29.…”

Section: Flow Cytometrymentioning

confidence: 99%

Bispecific Antibodies and Antibody–Drug Conjugates (ADCs) Bridging HER2 and Prolactin Receptor Improve Efficacy of HER2 ADCs

Andreev

Thambi

Bay

et al. 2017

Molecular Cancer Therapeutics

124

View full text Add to dashboard Cite

The properties of cell surface proteins targeted by antibodydrug conjugates (ADCs) have not been fully exploited; of particular importance are the rate of internalization and the route of intracellular trafficking. In this study, we compared the trafficking of HER2, which is the target of the clinically approved ADC ado-trastuzumab emtansine (T-DM1), with that of prolactin receptor (PRLR), another potential target in breast cancer. In contrast to HER2, we found that PRLR is rapidly and constitutively internalized, and traffics efficiently to lysosomes, where it is degraded. The PRLR cytoplasmic domain is necessary to promote rapid internalization and degradation, and when transferred to HER2, enhances HER2 degradation. In accordance with these findings, low levels of cell surface PRLR ($30,000 surface receptors per cell) are sufficient to mediate effective killing by PRLR ADC, whereas cell killing by HER2 ADC requires higher levels of cell surface HER2 ($10 6 surface receptors per cell). Noncovalently crosslinking HER2 to PRLR at the cell surface, using a bispecific antibody that binds to both receptors, dramatically enhances the degradation of HER2 as well as the cell killing activity of a noncompeting HER2 ADC. Furthermore, in breast cancer cells that coexpress HER2 and PRLR, a HER2xPRLR bispecific ADC kills more effectively than HER2 ADC. These results emphasize that intracellular trafficking of ADC targets is a key property for their activity and, further, that coupling an ADC target to a rapidly internalizing protein may be a useful approach to enhance internalization and cell killing activity of ADCs.

show abstract

Section: Flow Cytometrymentioning

confidence: 99%

Bispecific Antibodies and Antibody–Drug Conjugates (ADCs) Bridging HER2 and Prolactin Receptor Improve Efficacy of HER2 ADCs

Andreev

Thambi

Bay

et al. 2017

Molecular Cancer Therapeutics

124

View full text Add to dashboard Cite

show abstract

“…MTT assay is not reliable for the drugs with an action on mitochondria 34. And, most importantly, MTT assay relates to cytostatic effect, neither of cytotoxicity, apoptosis nor viability of the treated cells 35, 36. Some cells could remain perfectly viable with low metabolic activity and it may cause “fake” data 37…”

Section: Discussionmentioning

confidence: 99%

DeepScreen: An Accurate, Rapid, and Anti‐Interference Screening Approach for Nanoformulated Medication by Deep Learning

et al. 2018

View full text Add to dashboard Cite

Accuracy of current efficacy judgment methods for nanoformulated drug remains unstable due to the interference of nanocarriers. Herein, DeepScreen, a drug screening system utilizing convolutional neural network based on flow cytomerty single‐cell images, is introduced. Compared to existing experimental approaches, the high‐throughput system has superior precision, rapidity, and anti‐interference, and is cost‐cutting with high accuracy. First, it can resist most disturbances from manual factors of complicated evaluation progress. In addition, class activation maps generated from DeepScreen indicate that it may identify and locate the tiny variation from cell apoptosis and slight changes of cellular period caused by drug or even nanoformulated drug action at very early stages. More importantly, the excellent performance of assessment on two types of nanoformulations and fluorescent drug proves the fine generality and anti‐interference of this novel system. All these privileged performances make DeepScreen a very smart and promising system for drug detection.

show abstract

“…Compared with the traditional methods, this assay is more efficient, more reliable and faster. However, the employed methodology has limitations (7,24). It measures metabolic activity (mitochondrial function of PDLF), not necessarily the viability and cell proliferation (25).…”

Section: Discussionmentioning

confidence: 99%

Effect of Milk Renewal on Cell Viability In Vitro at Different Time Frames

et al. 2017

View full text Add to dashboard Cite

The purpose of this study was to evaluate if the renewal of milk as a storage medium, every 12, 24 and 48 h, is able to increase its ability to maintain human periodontal ligament fibroblasts (PDLF) viability over time. PDLF were soaked in Minimum Essential Medium at 37 °C (MEM-37) (positive control), tap water (Water) (negative control) and in skimmed milk (44 wells) at 5 °C and 20 °C. The skimmed milk was renewed every 12 h (Milk-12), 24 h (Milk-24) and 48 h (Milk-48) in 11 wells of each plate, and the milk in the remaining 11 wells of each plate was maintained in situ (not renewed milk) (NRM). After 24, 48, 72, 96 and 120 h, cell viability was determined by the tetrazolium salt-based colorimetric (MTT) assay. Data were statistically analyzed by Kruskal-Wallis, Scheffé and Mann-Whitney tests (α=5%). At 5 °C, only Milk-48 was significantly better than NRM. At 20 °C, NRM was more effective than Milk-12 and Milk-24 in all time periods. In relation to the temperature (5 °C or 20 °C), renewal of milk at 5 °C was better in maintaining cell viability than the renewal at 20 °C. In conclusion, the renewal of milk was able to increase its ability to maintain cell viability only when performed every 48 h in milk maintained at 5 °C.

show abstract

A Simple High-Content Cell Cycle Assay Reveals Frequent Discrepancies between Cell Number and ATP and MTS Proliferation Assays

Cited by 145 publications

References 43 publications

Bispecific Antibodies and Antibody–Drug Conjugates (ADCs) Bridging HER2 and Prolactin Receptor Improve Efficacy of HER2 ADCs

Bispecific Antibodies and Antibody–Drug Conjugates (ADCs) Bridging HER2 and Prolactin Receptor Improve Efficacy of HER2 ADCs

DeepScreen: An Accurate, Rapid, and Anti‐Interference Screening Approach for Nanoformulated Medication by Deep Learning

Effect of Milk Renewal on Cell Viability In Vitro at Different Time Frames

Contact Info

Product

Resources

About