Pe conjugated anti ccr1

RPMI 1640 was purchased from Life Technologies Corporation (Grand Island, NY, USA), containing 4.5 g/L D-Glucose, L-Glutamine and 110 mg/L Sodium Pyruvate. GM-CSF, IL-4 and TNF-α were purchased from Peprotech Asia (Revohot, Israel). Lipopolysaccharide (LPS) was purchased from Sigma Aldrich (Steinheim, Sweden). D-luciferin was purchased from Promega Corporation (Madison, WI, USA). The following antibodies were used for FACS analysis: fluorescein isothiocyanate (FITC)-conjugated anti-CD86, FITC-conjugated anti-I-A[b], FITC-conjugated anti-H-2Kb, Phycoerythrin (PE)-conjugated anti-CCR7, PE-conjugated anti-mouse CD40, PE-conjugated anti-CD11c and FITC conjugated anti-CD11c were purchased from BD PharMingen (SanDiego, CA). PE-conjugated anti-H-2Kb (SIINFEKL) was purchased from BioLegend (San Diego, CA). PE-conjugated anti-CCR1, PE-conjugated anti-CCR2, PE-conjugated anti-CXCR1 and PE-conjugated anti-CCR7 were purchased from R&D Systems (Minneapolis, MN, USA). All the isotype control antibodies were purchased from the same company as the detecting antibodies.

To analyze chemokine receptor expression of non-hematopoietic cells, cultured primary epidermal keratinocytes, dermal fibroblasts and dermal microvascular endothelial cells from different donors were analyzed using flow cytometry and the following monoclonal antibodies (mAb): PE-conjugated anti-CCR1 (53,504.111, IgG2β), CCR2 (48,607.211, IgG2β), CCR3 (61,828.111, IgG2α), CCR7 (150,503, IgG2α), CCR9 (112,509, IgG2α), CXCR4 (12G5, IgG2α), CXCR5 (51,505, IgG2α), CXCR6/STRL33 (56,811, IgG2β) all R&D Systems, Mineapolis, MN. CCR4 (1G1.1, IgG1), CCR5 (2D7/CCR5, IgG2α), CCR6 (11A9, IgG1) CXCR1 (5A12, IgG2β), CXCR2 (6C6, IgG1), CXCR3 (1C6/CXCR3, IgG1) all Pharmingen, San Diego, CA, CCR8 (Goat, IgG) Alexis Biochemicals, Grünberg, Germany, CCR10 (1908, IgG1) DNAX Research Inc., Palo Alto CA (or appropriate isotype controls (Pharmingen, San Diego, CA and R&D Systems, Mineapolis, MN, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). Briefly, 10⁶ cells were stained with anti-chemokine receptor mAb or isotype and analyzed using a FACScan and CELLQuest software (Becton Dickinson, San Jose, CA).