Human cxcl12 sdf 1 alpha quantikine elisa kit

SDF-1α was measured in CM from MCF-7 and MDA-MB-231 cells using a commercially available ELISA Kit in accordance with the instructions by the manufacturer (Human CXCL12/SDF-1 alpha Quantikine ELISA Kit, R&D Systems, Inc. Minneapolis, USA).
For binding assay, breast cancer cells were untreated (-) or treated with BRL 10 μM in phenol red-free media containing 5% CT-FBS for 24 h. Then, cells were harvested with versene reagent, washed twice in PBS and 10³ cells/well were incubated with CAF-CM in a final volume of 100 μl binding buffer (50 mM HEPES, pH 7.4, 1 mM CaCl2, 150 mM NaCl, 5 mM MgCl2, 5% bovine serum albumin). Samples were incubated for 60 min at 4°C with rotation. After incubation, cells were centrifuged and washed twice with 300 μl wash buffer (50 mM HEPES, pH 7.4, 1 mM CaCl2, 500 mM NaCl, 5 mM MgCl2) and freezed to −20°C and thawed to room temperature 3 times and then centrifuged at 1500×g for 10 minutes at 2 − 8°C to remove cellular debris. The supernatants were collected for assaying human SDF-1α levels (R&D Systems). The optical density of each well was determined using a microplate reader at 450 nm (Bio-Rad Model 3550 microplate reader, Richmond, CA) and normalized for cell number. At least three independent experiments were performed.

ELISA kits were used to measure plasma levels of (C-term) SDF-1α (Human CXCL12/SDF-1 alpha Quantikine ELISA Kit by R&D system), IL-6 (Human IL-6 ELISA Pro Kit by Mabtech, Nacka Strand, Sweden) and TNF-α (TNF alpha Ultrasensitive ELISA Kit, Thermo Fisher Scientific, Waltham, MA U.S.A.) following manufacturer’s instruction. Intra- and inter-assay coefficients of variation were 3.5 and 10.6% for SDF-1α, 2.5 and 3.8% for IL-6 and 6.0 and 8.5% for TNF-α, respectively. Cytokine/chemokine concentrations were calculated in duplicate by using a standard curve generated by serially diluting reconstituted standards and by measuring the absorbance at 450 nm in a microplate reader (Multiskan™ FC Microplate Photometer, Thermo Scientific).
Serum levels of extended range C-reactive protein were determined using the particle-enhanced turbidimetric immunoassay (PETIA technique; Siemens Dimension, USA). The reference range in this method is 0.0–5.0 mg/l. BNP levels were assessed using an Alere Triage reagent pack (Alere Inc., Ottawa, ON, CAN) and analysed in an automated D × I 800 immunoanalyzer (Beckman-Coulter, Fullerton, CA, USA).
Total GLP-1 was measured by a magnetic bead kit (Merck-Millipore, Vimodrone, Italy) according to kit instructions and analyzed on a MagPix (Luminex Corporation). The assay is based on the attachment of GLP-1 to magnetic beads and processing using LED excitation.