Mouse SDF-1 α / CXCL12 α ELISA Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-Bring all reagents and samples to room temperature (18–25 °C) before use.	-Wash by filling each well with Wash Buffer (300 µl) using a multi-channel Pipette or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels. -Read at 450 nm immediately after adding STOP solution.

Upstream tips
-Bring all reagents and samples to room temperature (18–25 °C) before use.
Protocol tips
-Wash by filling each well with Wash Buffer (300 µl) using a multi-channel Pipette or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels. -Read at 450 nm immediately after adding STOP solution.

Publication protocol

A total of 10,000 dispersed mouse CSMCs (n=3 per group) were seeded per well in a 6-well plate containing DMEM with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin and 2.5 µg/ml amphotericin B. Cells were incubated in a humidified incubator with 95% air and 5% CO2 at 37°C for 24 h with LPS (1 µg/ml) and varying amounts of metformin (0, 5, 10 and 20 mM). Following the incubation period treated samples were centrifuged at 350 × g for 5 min at 4°C. Conditioned media was stored at −20°C for further analysis and cells pellets were lysed immediately. Cell lysates were prepared using BashingBeads Lysis tubes from Zymo Research Corp. (Irvine, CA, USA) and cell lysis buffer containing protease inhibitor cocktail provided the a whole cell extraction kit (Abcam; cat. no. ab113475), according to the manufacturer's protocol. A nuclear protein extraction kit (Abcam; cat. no. ab113474) was used to extract total nuclear proteins from another set of control and treated samples. Lysates were centrifuged for 10 min at 10,000 × g at 4°C and supernatants were collected for further analysis. Total protein concentration of supernatants was measured using the DC protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Protein concentration was adjusted to 100 µg/ml in all samples. Protein levels of specific cytokines were evaluated by ELISA assay. Specific ELISA kits for TNF-α, IL-1α, M-CSF, TCA-3, SDF-1 and nuclear NF-κB p65 (pS536) were used to measure cytokine levels in lysates and conditioned media for control and treated samples according to the manufacturer's protocols.

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Manufacturer protocol

Download the product protocol from Sigma-Aldrich for Mouse SDF-1 α / CXCL12 α ELISA Kit below.

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