The CSF1R SRE Reporter Kit is designed for monitoring the activity of the CSF1R signaling pathway in cultured cells. The kit contains a transfection-ready vector for CSF1R and SRE luciferase reporter vector. Upon ligand binding, active CSF1R will initiate the MAPK/ERK signaling pathway, leading to expression of the SRE-controlled reporter. This reporter contains the firefly luciferase gene under the control of multimerized SRE responsive elements located upstream of a minimal promoter. The SRE reporter is premixed with a constitutively-expressing Renilla luciferase vector that serves as an internal control for transfection efficiency.

The kit also includes a non-inducible firefly luciferase vector premixed with constitutively- expressing Renilla luciferase vector as a negative control. The non-inducible luciferase vector contains the firefly luciferase gene under the control of a minimal promoter, without any additional response elements. The negative control is critical for determining pathway-specific effects and the background luciferase activity.

• Mammalian cell line and appropriate cell culture medium

• 96-well tissue culture plate or 96-well tissue culture-treated white clear-bottom assay plate

• Transfection reagent for mammalian cell line [We use Lipofectamine™ 2000 (Invitrogen #11668027) + Opti-MEM I Reduced Serum Medium (Invitrogen #31985-062). However, other transfection reagents work equally well.]

• Recombinant Human CSF1 (BPS Bioscience, #90215-B) or Recombinant Human IL34 (Biolegend, #577904)

• Low serum (0.5% FBS) growth medium optimized for parental cell type

For HEK293 cell lines use

BPS Medium 8 (BPS Bioscience, #79385): MEM medium (Hyclone #SH30024.01) supplemented with 0.5% FBS (Invitrogen #26140-079), 1% non-essential amino acids (Hyclone #SH30238.01), 1 mM Na pyruvate (Hyclone #SH30239.01), 1% Penicillin/Streptomycin (Hyclone SV30010.01)

• Dual Luciferase Assay System:

Dual Luciferase (Firefly-Renilla) Assay System (BPS Bioscience, #60683): This system assays cells directly in growth medium. It can be used with any luminometer. Automated injectors are not required.

• Luminometer

FMS, CD115

Each kit contains sufficient reagents for 500 assays using a 96-well plate.

See assay kit data sheet for detailed protocol.

Store cell line in liquid nitrogen immediately upon receipt. Store other components as described below. Stable for at least 6 months from date of receipt when stored as directed.

• Monitor CSF1R/MAPK/ERK signaling pathway activity and SRF-mediated activity.

• Screen for activators or inhibitors of the CSF1R/MAPK/ERK signaling pathway.

• Study effects of RNAi or gene overexpression.

1. Cannarile, Michael A., et al. “Colony-Stimulating Factor 1 Receptor (CSF1R) Inhibitors in Cancer Therapy.” J. Immunother. Cancer, 5(1): 53 (2017)

2. Liu, Yang, and Xuetao Cao. “The Origin and Function of Tumor-Associated Macrophages.” Cellular Molec. Immunology, 12(1): 1–4 (2014).

3. Luo, Jian, et al. “Colony-Stimulating Factor 1 Receptor (CSF1R) Signaling in Injured Neurons Facilitates Protection and Survival.” J. Exp. Med., 210(1): 157–172 (2013).

4. Yao, G.-Q., et al. “CSF-1 Induces Fos Gene Transcription and Activates the Transcription Factor Elk-1 in Mature Osteoclasts.” Calc. Tissue Int., 76(5): 371–378 (2005).

Avoid freeze/thaw cycles.

Immunotherapy

Cell-Based Assays