R kit from Stratagene and by following the manufacturers instructions. Subsequently, RT PCR was performed under standard conditions using selleck kinase inhibitor primers specific for CCR1, CCR2 and GAPDH. The primer sequences used here were The annealing temperature used for RT PCR was 55 C for 30 seconds and the e tension temperature was 72 C for 1 minute. typically 30 cycles of PCR were performed. Under these conditions the product sizes for CCR1, CCR2 and GAPDH were 567 bp, 580 bp and 420 bp respectively. Antibody staining and FACS analysis THP 1 cells or PBMCs were resuspended in ice cold stain ing buffer and incu bated with Fc block for 5 minutes at 4 C. Subsequently, primary antibodies were added at a final concentration of 0. 5 g l. The cells were then incubated at 4 C for 25 minutes, after which time they were washed twice in staining buffer.
The secondary antibody used for these e periments was Ale a 488 at a final concentration of 1 g l. This time the cells were incubated at 4 C for 25 minutes in the dark. Following incubation with the secondary anti body, the cells were again washed twice, and then resus pended in 500 l of staining buffer. Samples were finally analyzed on a FACScan flow cytometer using Cellquest 3. 2. 1f1 software. Peripheral blood monocytes, monocyte derived macrophages and THP 1 cells were also stained for CD36, CD11b and CD68. Transient transfection using DEAE De tran THP 1 cells, grown to a density of 5 8 105 ml, were resuspended in Tris buffered saline. THP 1 cells were then added to 1 ml of TBS containing 5 g of the CCR2 promoter luciferase construct, 2 g of the renilla control construct and 500 g ml DEAE De tran.
This mi ture was then left at room temperature for one hour. Ne t, DMSO was added to the cells drop wise to a final concen tration of 10% and incubated for 2 minutes at room tem perature. Subsequently, the cells were washed twice in TBS, once in RPMI 1640 medium lacking FCS and antibi otics and once in RPMI 1640 complete medium. The cells were then resuspended in RPMI 1640 complete medium, stimulated with PMA and ionomycin and finally incubated at 37 C and 5% CO2 for 48 hours. After the 48 hour incubation period, cell e tracts were made using the luciferase reporter lysis buffer. Each lysate was subsequently assayed in the dual luci ferase reporter assay following the manufac turers instructions.
Luciferase activity was determined using a Monolight series 2010 luminometer and then normalized Drug_discovery to the renilla control. Results Freshly isolated monocytes selectively sellectchem downregulate CCR2, but not CCR1, in culture Human monocytes were isolated from blood leukopacks and placed in culture for up to 5 days. During this time these cells underwent changes in both morphol ogy and gene e pression. Freshly isolated monocytes ini tially appeared small and round, but after 5 days in culture they became adherent, and increased in both size and granularity. Ne t, we analyzed changes in the e pression of the macrophage differentiation markers CD11b,