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Human M-CSFR Reporter Assay Kit

SIZE SKU PRICE
1 x-384 well format assays—
1 x-96 well format assays—
SIZE SKU
1 x-384 well format assays
1 x-96 well format assays
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Product Description and Product Data

This is an all-inclusive cell-based luciferase reporter assay kit targeting the the Human Macrophage Colony-Stimulating Factor Receptor (M-CSFR). INDIGO’s M-CSFR reporter assay utilizes proprietary mammalian cells that have been engineered to provide constitutive expression of the M-CSFR. In addition to M-CSFR Reporter Cells, this kit provides two optimized media for use during cell culture and in diluting the user’s test samples, a reference agonist, Luciferase Detection Reagent, and a cell culture-ready assay plate. The principal application of this assay is in the screening of test samples to quantify any functional activity, either agonist or antagonist, that they may exert against M-CSFR. This kit provides researchers with clear, reproducible results, exceptional cell viability post-thaw, and consistent results lot to lot. Kits must be stored at -80C. Do not store in liquid nitrogen. Note: reporter cells cannot be refrozen or maintained in extended culture.

Features

  • Clear, Reproducible Results

  • All-Inclusive Assay Systems
  • Exceptional Cell Viability Post-Thaw
  • Consistent Results Lot to Lot

Product Specifications

Target TypeCytokine Receptor, Growth Factor Receptor
SpeciesHuman
Receptor FormHybrid
Assay ModeAgonist, Antagonist
Kit Components
  • Human M-CSFR Reporter Cells
  • Cell Recovery Medium (CRM)
  • Compound Screening Medium (CSM)
  • M-CSF, (ref. agonist; in PBS/0.1%BSA)
  • Detection Substrate
  • Detection Buffer
  • White, sterile, cell-culture ready assay plate
Shelf Life6 months
Shipping RequirementsDry Ice
Storage temperature-80C

Data

M-CSFR activation dose response analyses. Activation dose-response assays were performed according to the protocol provided in the Technical Manual. 200 ul / well of M-CSFR Reporter Cell suspension was dispensed into the 96-well assay plate, which was then incubated for 4 hours. The concentrated stock of M-CSF (provided) was further diluted using CSM to produce treatment media at the desired assay concentrations. The pre-culture media were discarded from the assay wells and 200 ul per well of the prepared treatment media were dispensed (n = 3/conc.), including ‘untreated control’ wells. Following 22 hours incubation the treatment media were discarded, Luciferase Detection Reagent was added, and luminescence intensity per well was quantified. Values of average relative light units (RLU) and corresponding values of standard deviation (SD), percent coefficient of variation (%CV), Fold-Activation and Z’ were determined for each treatment concentration. GraphPad Prism software was used to perform the least-squares method of non-linear regression to plot Fold Activation vs. Log10[ng/mL] and to determine EC50 values.
M-CSFR Inhibition dose-response analyses. M-CSFR reporter cells were co-treated with an EC80 concentration of the reference activator M-CSF (provided) and varying concentrations of the small molecule M-CSFR inhibitors Edicotinib, TPX-002, PLX5622 and Sulfatinib. The range of determined IC50 values is shown; (n = 3/conc.). INDIGO’s Live Cell Multiplex (LCM) Assay was performed to confirm that no treatment concentrations were cytotoxic (data not shown). Non-linear regression analyses of RLU vs. Log10[Inhibitor, nM] were plotted and IC50 determinations were made using GraphPad Prism software. All compounds were obtained from Cayman Chemical, Ann Arbor MI, USA.

Target Background

Macrophage colony-stimulating factor (M-CSF) is a cytokine expressed in a wide range of cells and tissues. It stimulates progenitor cells from bone marrow and plays a role in the development, proliferation, and maintenance of phagocytes like monocytes, dendritic cells and microglia.

The activity of M-CSF is mediated through binding interactions with the M-CSF Receptor (M-CSFR; also known as CSF1R). M-CSF/M-CSFR activity is involved in various pathologies such as ovarian cancer, breast cancer, rheumatoid arthritis, and cutaneous lupus. Recent phase II clinical trials have indicated that monoclonal antibody or small molecule antagonists targeting M-CSFR have reduced the inflammatory response often associated with rheumatoid arthritis.

In the brain, M-CSF is secreted by neurons, astrocytes and microglia and is involved in brain development. M-CSF-deficient animals have severe brain deficits with abnormalities associated with the cerebral cortex. Other phenotypic traits associated with M-CSF deficiency include reduced body weight and skeletal defects1. Additionally, in many neurodegenerative diseases, microglia are becoming a key target for therapeutic purposes as they eliminate toxic elements from the brain and set the conditions for repair and remyelination1. Low level M-CSF/M-CSFR expression or receptor inhibition in microglia has been associated with pre-symptomatic Alzheimer’s disease (AD), Parkinson’s disease (PD), multiple sclerosis (MS) and frontotemporal dementia. Consequently, M-CSF and its specific receptor, M-CSFR, command considerable interest in therapeutics development and drug safety screening.

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Macrophage Colony-Stimulating Factor Receptor (M-CSFR)

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