Mouse cxcl12 sdf 1 alpha quantikine elisa kit

Fibroblasts were isolated from the dorsal skin of WT mice as previously described.^{25 (link)} Fibroblasts were plated out on three 6-well plates (1.5 × 10⁵ cells/well) with high glucose (4.5 g/L) Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Fibroblasts were then exposed to high glucose medium in hypoxia (37°C, 1% oxygen) for 96 hours with serial medium changes occurring every 24 hours. The cells were then exposed to either MK0626 (10 μM) or high glucose media alone. Total protein was quantified using the Quick Start Bradford Protein Assay Kit (Bio-Rad, Hercules, California). SDF-1 protein levels were measured using the Mouse CXCL12/SDF-1 alpha Quantikine ELISA Kit (R&D Systems, Minneapolis, Minnesota).

Bone marrow extracellular fluid was obtained by flushing each femur and tibia with 1000 μL HBSS, and the supernatant was harvested after centrifugation at 400g for 5 minutes. CXCL12 protein quantification in bone marrow extracellular fluid was determined by ELISA (Mouse CXCL12/SDF-1 alpha Quantikine ELISA Kit; R&D System) according to the manufacturer's instructions.

Antibodies used for staining included: anti-pan-cytokeratin (clone C11, 1:500) and vimentin (clone D21H3, 1:200) (Cell Signaling), anti- α-smooth muscle actin (α-SMA) (clone 1A4, 1:200) (Dako), anti-Ki-67 (rabbit polyclonal, ab15580, 1:300) (Abcam), anti-CD105-PE (clone SN6, 1:100) (eBioscience), anti-mitochondria (clone 113-1, 1:100) and anti-fibroblast specific protein-1 (FSP-1; rabbit polyclonal, 07-2274, 1:200) (Millipore). IL-6 ELISA kit was purchased from BD Biosciences. CCL5 DuoSet ELISA kit and mouse CXCL12/SDF-1 alpha Quantikine ELISA Kit were from R&D Systems. Alexa Fluor® fluorescent dye-conjugated secondary antibodies were from Life Technologies for immunofluorescent staining.

Plasma CXCL12 levels were measured pre- and post-AMD3100 dosing for first and second doses in the first mouse cohort. Blood samples were spun down at 1700rpm for 15 mins and plasma extracted by pipetting. Plasma from each blood draw was frozen to allow simultaneous testing of all samples for CXCL12 level by ELISA assay using Mouse CXCL12/SDF-1 alpha Quantikine ELISA Kit (R+D Systems, MN, US) with analysis of optical density carried out on the Epoch microplate spectrophotometer (Biotek, VT, US).