Standard protocol for the PIGRET assay, a high-throughput reticulocyte Pig-a assay with an immunomagnetic separation, used in the interlaboratory trial organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen and Genome Society

AbstractThe PIGRET assay is one of the Pig-a assays targeting reticulocytes (RETs), an in vivo genotoxicity evaluation method using flow cytometry with endogenous reporter glycosylphosphatidylinositol anchor protein. The PIGRET assay with RETs selectively enriched with anti-CD71 antibodies has several desirable features: high-throughput assay system, low background frequency of mutant cells, and early detection of mutation. To verify the potential and usefulness of the PIGRET assay for short-term testing, an interlaboratory trial involving 16 laboratories organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen and Genome Society was conducted. The collaborating laboratories assessed the mutagenicities of a total of 24 chemicals in rats using a single-treatment design and standard protocols for conducting the Pig-a assay on the total red blood cell assay and the PIGRET assay. Here the standard protocol for the PIGRET assay was described in detail.

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Standard protocol for the total red blood cell Pig-a assay used in the interlaboratory trial organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen Society

Genes and Environment ◽

10.1186/s41021-019-0121-z ◽

2019 ◽

Vol 41 (1) ◽

Cited By ~ 1

Author(s):

Satsuki Chikura ◽

Takafumi Kimoto ◽

Satoru Itoh ◽

Hisakazu Sanada ◽

Shigeharu Muto ◽

...

Keyword(s):

Blood Cell ◽

Red Blood Cell ◽

Study Group ◽

Standard Protocol ◽

Environmental Mutagen ◽

Interlaboratory Trial

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In vitro to in vivo concordance of a high throughput assay of bone marrow toxicity across a diverse set of drug candidates

Toxicology Letters ◽

10.1016/j.toxlet.2009.03.012 ◽

2009 ◽

Vol 188 (2) ◽

pp. 98-103 ◽

Cited By ~ 22

Author(s):

Andrew J. Olaharski ◽

Hirdesh Uppal ◽

Matthew Cooper ◽

Stefan Platz ◽

Tanja S. Zabka ◽

...

Keyword(s):

Bone Marrow ◽

High Throughput ◽

Bone Marrow Toxicity ◽

Drug Candidates ◽

High Throughput Assay

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Semi-automated high-throughput substrate screening assay for nucleoside kinases

10.33774/chemrxiv-2021-k0w7q ◽

2021 ◽

Author(s):

Katja Hellendahl ◽

Maryke Fehlau ◽

Sebastian Hans ◽

Peter Neubauer ◽

Anke Kurreck

Keyword(s):

High Throughput ◽

Viral Infections ◽

Screening Assay ◽

Liquid Handling ◽

Wide Range ◽

High Throughput Assay ◽

Liquid Handling Robot

Nucleoside kinases (NKs) are key enzymes involved in the in vivo phosphorylation of nucleoside analogues used as drugs to treat cancer or viral infections. Having different specificities, the characterization of NKs is essential for drug design and the production of nucleotide analogues in an in vitro enzymatic process. Therefore, a fast and reliable substrate screening assay for NKs is of great importance. Here, we report the validation of a well-known luciferase-based assay for the detection of NK activity in 96-well plate format. The assay was semi-automated using a liquid handling robot. A good linearity was demonstrated (r² >0.98) in the range of 0 to 500 µM ATP, and it was shown that also alternative phosphate donors like dATP or CTP were accepted by the luciferase. The developed high-throughput assay revealed comparable results to HPLC analysis. The assay was exemplary used for the comparison of the substrate spectra of four nucleoside kinases using 20 (8 natural and 12 modified) substrates. The screening results correlated well with literature data and, additionally, previously unknown substrates were identified for three of the NKs studied. Our results demonstrate that the developed semi-automated high-throughput assay is suitable to identify best performing NKs for a wide range of substrates.

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Generation of endogenous pH-sensitive EGF receptor and its application in high-throughput screening for proteins involved in clathrin-mediated endocytosis

eLife ◽

10.7554/elife.46135 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 4

Author(s):

Mads Breum Larsen ◽

Mireia Perez Verdaguer ◽

Brigitte F Schmidt ◽

Marcel P Bruchez ◽

Simon C Watkins ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Gene Editing ◽

Egf Receptor ◽

Ph Sensitive ◽

Fluorescence Excitation ◽

High Throughput Analysis ◽

Egfr Expression ◽

High Throughput Assay

Previously we used gene-editing to label endogenous EGF receptor (EGFR) with GFP and demonstrate that picomolar concentrations of EGFR ligand drive signaling and endocytosis of EGFR in tumors in vivo (Pinilla-Macua et al., 2017). We now use gene-editing to insert a fluorogen activating protein (FAP) in the EGFR extracellular domain. Binding of the tandem dye pair MG-Bis-SA to FAP-EGFR provides a ratiometric pH-sensitive model with dual fluorescence excitation and a single far-red emission. The excitation ratio of fluorescence intensities was demonstrated to faithfully report the fraction of FAP-EGFR located in acidic endosomal/lysosomal compartments. Coupling native FAP-EGFR expression with the high method sensitivity has allowed development of a high-throughput assay to measure the rates of clathrin-mediated FAP-EGFR endocytosis stimulated with physiological EGF concentrations. The assay was utilized to screen a phosphatase siRNA library. These studies highlight the utility of endogenous pH-sensitive FAP-receptor chimeras in high-throughput analysis of endocytosis.

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