Receptor binding assays

For the competition binding studies against CXCR4, a concentration range of Plerixafor was incubated for 3 hours at 4°C in binding buffer (PBS containing 5 mM MgCl2, 1 mM CaCl2, 0.25% BSA, pH 7.4) with 5 × 105 CCRF-CEM cells and 100 pM 125I-SDF-1α (2200 Ci/mmol) in Milipore DuraporeTM filter plates. Unbound 125I-SDF-1α was removed by washing with cold 50 mM HEPES, 0.5 M NaCl pH 7.4. The competition binding assay against BLT1 was performed on membranes from CHO-S cells expressing recombinant BLT1. The membranes were prepared by mechanical cell lysis followed by high speed centrifugation, re-suspended in 50 mm HEPES, 5 mM MgCl2 buffer and flash frozen. The membrane preparation was incubated with Plerixafor for 1 hour at room temperature in an assay mixture containing 50 mM Tris, pH 7.4, 10 mM MgCl2, 10 mM CaCl2, 4 nM LTB4 mixed with 1 nM 3H-LTB4 (195.0 Ci/mmol) and 8 μg membrane. The unbound 3H-LTB4 is separated by filtration on Millipore Type GF-C filter plates.

Cell lines

U2OS cells expressing EGFP-CXCR4

Preparation method

Limited solubility in DMSO. General tips for obtaining a higher concentration: Please warm the tube at 37℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reaction Conditions

2.5 mg/mL; 30 min

Applications

CXCR4 and SDF-1 were key factors in regulating cancer cell invasion and metastasis, and Plerixafor effectively prevented the binding of SDF-1 to CXCR4, inhibiting cancer metastasis.

Animal models

C57BL/6 mice with segmental bone defect

Dosage form

5 mg/kg; i.p.

Applications

Cohorts of mice were administered with PBS, IGF1, PDGF, SCF or VEGF for five consecutive days and Plerixafor on the 5th day. The number and size of the colonies were highest in mice injected with IGF1 and Plerixafor than those treated with PDGF, SCF or VEGF plus Plerixafor.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

References:

[1]. Fricker SP, Anastassov V, Cox J, Darkes MC, Grujic O, Idzan SR, Labrecque J, Lau G, Mosi RM, Nelson KL, Qin L, Santucci Z, Wong RS. Characterization of the molecular pharmacology of AMD3100: a specific antagonist of the G-protein coupled chemokine receptor, CXCR4. Biochem Pharmacol. 2006 Aug 28;72(5):588-96.

[2]. Li J, Oupick? D. Effect of biodegradability on CXCR4 antagonism, transfection efficacy and antimetastatic activity of polymeric Plerixafor.Biomaterials. 2014 Jul;35(21):5572-9.

[3]. Kumar S, Ponnazhagan S. Mobilization of bone marrow mesenchymal stem cells in vivo augments bone healing in a mouse model of segmental bone defect. Bone. 2012 Apr;50(4):1012-8.