Innoprot

 
Innoprot社では、Stable cell line、Primary cellを用いた細胞ベースのアッセイサービスを中心に提供しています。
独自のProprietaryな技術を用いたHigh Content Screening (HCS)、Phenotypic Screening等の様々なアプローチにより、CNS研究、癌研究を中心にサポートします。

《サービス一覧》
 
《サービス概要》
◆ GPCR platform
 
  • Fluorescent GPCR Internalization Assay
    化合物のGPCR活性化もしくはインターナリゼーションを、蛍光強度およびその分布により評価します。
    <ターゲットリスト>
    ADRB2-tGFP/CHO-K1 CNR2-tGFP/CHO-K1 DRD5-tGFP/SH-SY5Y PAC1-tGFP/U2OS
    BDK2R-tGFP/CHO-K1 CRHR2-tGFP/CHO-K1 FPRL1-tGFP/U2OS S1P1-tGFP/CHO-K1
    BDKR1-tGFP/CHO-K1 CXCR2-tGFP/U2OS GLP2R-tGFP/CHO-K1 SSTR2-tGFP/U2OS
    CALCR1-tGFP/CHOK1 DRD1-tGFP/SH-SY5Y OPRD1-tGFP/HEPG2 SSTR3-tGFP/U2OS
    CCR2-tGFP/U2OS DRD2-tGFP/SH-SY5Y OPRK-tGFP/HEPG2 TACR1-tGFP/SH-SY5Y
    CHRM1-tGFP/CHO-K1 DRD3-tGFP/SH-SY5Y OPRS1-tGFP/CHO-K1 TACR3-tGFP/SH-SY5Y
 

Activation and Internalization assay for CCR2-tGFP (Ec50 =0.73 ng/ml)

   
Internalization of CCR2 stimulated with human MCP-1. Concentrations from 0 to 10 μg/ml were tested for 3h. Concentration response curve for human MCP-1 in CCR2 cell line.
 
  • NOMAD® High Content Screening (Ca++, cAMP, DAG Nomad® Biosensor)
    • Measure the GPCR activity in living cells using the same fluorescent backbone.
    • Measure the second messenger concentration changes involved in GPCR activation.
    • Robust and reproducible and could be combined with internalization studies.
   
 
 NOMAD® Biosensor - How it works  NOMAD® Biosensor - versions
When a receptor is activated, second messengers involved in the GPCR pathway vary their concentration. Upon GPCR activation, the increase in the second messenger concentration leads to a change in the structual folding of NOMAD® Biosensor that promotes its cellular relocation. The second messenger transduction protein binding peptide of the bionsensor could be replaced depending on the second messenger involved in the GPCR activation pathway, resulting three different versions of NOMAD® Biosensors.
 

Activation assay for TACR3 - Ca++ Nomad® cells (EC50 = 8.9x10-8 M)

   
Redistribution of TACR3 stimulated with Substance P. Concentrations from 0 to 10 μM were tested for 24h. Activation and redistribution processes were detected. Concentration response curve for Substance P in Ca++Nomad® TACR3 receptor cell line


Activation assay for GLP1R - cAMP Nomad® cells (EC50 = 1.1x10-6 M)

   
Redistribution of GLP1R stimulated with GLP1. Concentrations from 0 to 10 μM were tested for 48h. Activation and redistribution processes were detected. Concentration response curve for GLP1 in cAMP Nomad® GLP1R receptor cell line.
In vitro Disease Models (Cell Lines)
  Examples:
 

Activation and Internalization assay for CCR2-tGFP (Ec50 =0.73 ng/ml)

When APP processing is stopped by any compound, retained fluorescent APP spots are formed. Determination of IC50 values for APP processing inhibitor LP226A1.
In vitro Disease Models (Primary Cells)
  Examples:
 
  % Survival. Cell viability reduction in primary neuron cultures in response to increasing concentration of Aβ 1-40
◆ Phenotypic Screening platform (High Content Analysis)
 

プライマリーセルリスト (一例) (pdf)

  • Toxicity & Apoptosis
  • Organelle & trafficking
    • Nucleo-cytoplasmic translocation
    • Endosomal trafficking
    • Plasma membrane translocation
    • Membrane ruffling
    • Golgi integrity
    • Mitochondrial translocation
  • Transient transfections
    • Sub population analysis
  • Stem Cell Differentiation Studies
    • Adipogenesis
    • Osteogenesis
    • Chondrogenesis

  • Signalling pathways
    • Phospholipid sensors: Plasma membrane sensors
    • Phospholipid sensors: Endosomal sensors
    • Cell survival signalling
    • Cell migration signalling
    • Stress response signalling (pdf)
    • Reporter gene expression
    • Protein expression
  • Cell cycle
    • Cell cycle status reporting (automated)
    • DNA replication studies
    • Cell proliferation
    • Muliplexed cell cycle analysis
    • Cell cycle (DNA content)
  • Cell morphology & health
    • Neurite extension analysis
    • Angiogenesis Studies
    • Cell rounding
    • Measurement of discrete structures at cell surface
Cytotoxicity induced by drugs, radiation, oxidative stress, hyperosmolarity and other agents in ocular cell lines
Other parameters: nuclei number and nuclei size, cell shrinkage, LDH release, MTT-WST
Epithelial tight junction disruption in response to different stimuli
Induced by Hypoxia, cytokines, LPS and other reagents (BAK)
Cell lines: HCE, WKD, Primary Human Retinal Pigment Epithelial Cells
in vitro wound-healing assay
Wound healing evaluation from day 1 to 3 after injury
Cell lines: HCE, WKD, Primary Fibroblasts
End points: Wound closure rate over time, Cell Proliferation rate (Ki67 staining)
◆ Toxicity Assays
 

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