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Realization of brain well-being society through treatment of
intractable brain diseases and utilization of optimal brain functions

Evaluation system for in vitro efficacy & PoC

Cell culture systems

Evaluation method

  • 1Primary cell culture model
    • Primary neuron/astrocyte/microglia culture, Neuronal precursor cell culture Organotypic brain slice culture
  • 2Cell lines
    • HT22, HN3, Neuro2A, N27, SH-SY5Y, PC12, C6 glioma, BV-2, bEnd.3 cell


Evaluation method

  • 1 In vivo
    • Hematoxylin & Eosin staining, Cresyl violet staining
    • TUNEL stain, Fluoro Jade B stain
    • TTC staining
    • Immunohisto(cyto)chemistry
  • 2 in vitro
    • LDH, MTT, PI Staining, calcein AM
  • 3 Neuroprotectant: expression of BDNF, NT, GDNF etc (Western blot)


Evaluation method

  • 1 ROS
    • ROS measurement: DCFH2-DA, DHE, Rhodamine123, TMRE
    • 4-HNE/MDA, Peroxiredoxin
    • Antioxidant enzyme: GSH, SOD, NADPH Oxidase, HO-1 expression
    • Western blot, RT-PCR, Enzyme activity assay (ELISA)
  • 2 Inflammatory mediators
    • inflammatory Cytokines expression: iL-1beta, IL-6, IL-18, TNFalpha, TGFbeta
    • COX-2/PGs, iNOS/NO expression and release
    • mmps, tPA/PAI-1 expression and activity
    • Western blot, RT-PCR, Zymography, ELISA

Biomolecular change

Evaluation method

  • 1 Gene expression: RT-PCR, Microarray, PCR-Array, ChIP assay
  • 2 Gene knockdown by Transient transfection: siRNA/shRNA transfection
  • 3 Gene Overexpression

Glial activation

Evaluation method

  • 1 GFAP, NG2, Iba-1, OX-6, MBP, Ki-67 staining
  • 2 migration assay : migration chamber, Co-culture system (transwell )
  • 3 phagocytosis: phagocytotic receptor (Western blot), phagocytosis
  • 4 protease activity: MMP9, tPA/PAI-1 activity and expression

Synaptic plasticity and synaptogenesis

Evaluation method

  • 1 NCAM, BDNF, tPA/PAI-1, MMPs expression and release (Western blot, RT-PCR, ELISA, Zymography)
  • 2 PSD95, Synaptophysin expression (Immunohistochemistry, Western blot)
  • 2 nNOS/NO activity
  • 2 synapse: Synaptosomal fraction
  • 2 EM analysis

Proliferation and differentiation of Neural stem cell

Evaluation method

  • 1 Proliferation: MTT, ICC (Ki-67, BrdU, Nestin, doublecortin)
  • 2 Differentiation: NeuN/Tuj-1, Nestin, GFAP (ICC/IHC, Western blot)
  • 3 NCAM, BDNF, tPA/PAI-1, MMPs Expression, release and activity (Western blot, RT-PCR, ELISA, tPA/PAI-1 Zymography)
  • 4 Live cell imaging
  • 5 Neurite outgrowth Inucyte analyzing system, neurite number and neurite length

Mitochondrial function

Evaluation method

  • 1 OCR: Seahorse XF Cell Mito Stress Analyzer system
  • 2 Morphological change: Fusion/Fission, ICC, EM
  • 3 ROS: mitoSOX/Mitotracker imaging

Signal transduction

Evaluation method

  • 1 Signaling molecules related with inflammation and cell death
    • Src, Fyn, Syk, calcineurin, TLR2/4, JAK/STAT, MAPKs, PKC, PKA, PKG, iNOS, mTOR, s6k1, AMPK, PI3K/Akt/GSK3beta, NF-kB, IkB, AP1, Nrf2/Keap1, FOXO etc
  • 2 Synapse, neurogenesis and cognition
    • PI3K/Akt, MAPKs, BDNF, GDNF, eNOS/nNOS, c-fos, ARC, CREB, CAMKII, Casein kinase, aurora kinase etc

Ca2+ Imaging

Evaluation method

  • 1 Ca2+ imaging: Fura2/4 AM
  • 2 Oregon Green BAPTA-1
  • 3 ELISA/Immunofluorescence

Epigenetic modulation

Evaluation method

  • 1 DNA methylation : DNA methyltransferases (DNMT1, 3a/3b, MeCP2
  • 2 Histone acetylation: Histone acetyltransferase (HATs), Histone deacetylase (HDACs)
  • 3 Histone methyltransferases (HMTs), histone demethylases, and histone kinases
  • 4 Enzyme expression : Western blot/RT-PCR
  • 5 HDAC activity assay
  • 6 DNA methylation assay (ChiP assay)

Neurotransmitter change

Evaluation method

  • 1Fast scan cyclic voltammetry
  • 2 Functional reuptake assay (NET, SERT, DAT)

Discovery of New target

Evaluation method

  • 1 Microarray, PCR Array, Real time-PCR