MK-0812

Kinase experiment:

Human whole blood is collected in EDTA tubes and used within 1 h of blood collection. For antagonist treated samples, blood (200 ?L) is pre-incubated with MK-0812 (0.1% final DMSO concentration) for 30 min at room temperature. After which, 20 ?L of FITC conjugated anti-CD14 antibody and 4 ?L of chemokine or buffer is added to each sample and mixed lightly. An aliquot (100 ?L) of the blood mixture is incubated for 10 min at 37°C, immediately placed on ice and lightly fixed with 250 ?L of ice cold fixative (49 mL PBS, 1.0 mL 4% para-formaldehyde) for 1 min. Red blood cells are lysed by adding 1.0 mL of ice cold lysis solution (0.15 M NH4Cl2, 10 mM sodium bicarbonate, and 1 mM EDTA), and incubated for 20 min on ice. After complete lysis of red blood cells, 100 ?L of 4% para-formaldehyde is added and the samples are analyzed by flow cytometry for forward scatter measurements[1].

Animal experiment:

Mice[2] Female BALB/c mice are used between 8 and 10 weeks of age. SCH563705 or MK0812 are administered in a 0.4% MC solution by 30 mg/kg oral gavage (p.o.). Two hours later, the frequency of CD11b+Ly6G-Ly6Chi monocytes and CD11b+Ly6G+Ly6C+ neutrophils is determined by flow cytometry.

References:

[1]. Wisniewski T, et al. Assessment of chemokine receptor function on monocytes in whole blood: In vitro and ex vivo evaluations of a CCR2 antagonist.J Immunol Methods. 2010 Jan 31;352(1-2):101-10.
[2]. Min SH, et al. Pharmacological targeting reveals distinct roles for CXCR2/CXCR1 and CCR2 in a mouse model of arthritis.Biochem Biophys Res Commun. 2010 Jan 1;391(1):1080-6.