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Ca2+- Flux Inhibition Fluorescence Assay in Jurkat Cells
Example for Ion Channels Inhibitors Screening

  • An increase in intracellular free Ca2+ concentration is a ubiquitous signalling mechanism that regulates a broad spectrum of cellular processes and is generally store-dependent.
  • Store-operated Ca2+ release-activated Ca2+ channels (CRAC) are very high Ca2+ selective channels composed of two protein subunits: STIM1 Ca2+sensor protein placed in the ER and ORAI1 Ca2+ channel protein in plasma membrane.
  • In T cells, activation of CRAC leads to short term effects (reduced lymphocyte motility) and long term effects (altered gene expression & production of cytokines, ex. IL-2 and IFN-γ).
  • Therefore, inhibition of CRAC (Icrac) is expected to modulate T cell activation and channels are potential target for new anti-inflammatory drugs as RA and psoriasis.
  • Ca2+ -flux inhibition fluorescence assay in Jurkat cells is useful 384-plate format HTS screening assay for quick identification of STIM1/ORAI1 inhibitory compounds what is representative for CRAC inhibition in T cells.
  • The assay has excellent correlation with patch clamp electrophysiology assay.

ASSAY PRINCIPLE

Intracellular Ca2+ is measured with Ca2+ sensitive dye.
Binding with Ca2+ ions  results in fluorescent signal

Parameter
Ca-flux inhibition
Z’
>=0.5
Window (S/B)
Reproducible value
R2
>=0.8
Hill slope
0.5-2
IC50
Deviation=<3x
Range (Top-bottom)
Reproducible value (~30%-70%)

Assay results and data analysis

Results are presented in a form of graphs and tables, with calculated IC50 values  in nM.
Method is programmed for PE Janus robotic system in WinPrep for Janus pipetting software.
Control compound, YM-58483/BPT2 (Astellas), a potent inhibitor of CRAC channels, blocking thapsigargin-induced sustained calcium influx, is tested on every plate.
Calculation of IC50 data, curves and QC analysis is made by using Excel tools and GraphPadPrism software.
QC criteria parameters (Z’, S:B, R2, HillSlope) were checked for every IC50 curve.

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