Recombinant human cxcl12 protein

Transmigrations were conducted with AP48 48-well Boyden Chambers (Neuro Probe, Inc. USA) according to the protocols that are provided by the manufacturer. The chemotaxis chambers had a 5 mm diameter polycarbonate film with 5 µm pore. We seeded 2×10⁴ of lymphocytes into the top wells. To induce the cell migration, a recombinant human CXCL12 protein (R&D Systems, USA) with a broad concentration range from 0 ng/ml to 1000 ng/ml was added into the lower chambers. CXCR4-specific antagonist. AMD3100 (Sigma, USA) was used to inhibit the induced CXCL12-binding transmigration. AMD3100 was dissolved in dimethylsulfoxide (DMSO) (Beijing Solarbio S&T Co., Ltd., China mainland) to prepare 1 mg/ml of stock concentration and was stored at 4°C. The inhibitory effect of AMD3100 was tested at the concentrations ranging from 0.1 µg/ml to 100 µg/ml.

HCC cells were seeded into 6-well cell culture plates at a concentration of 1 × 10⁶ cells/well and incubated for 24 h. Then, the confluent monolayer of cells was scratched with a 200-µl pipette tip and then washed twice with 1× PBS. Next, 2 mL DMEM containing 2% FBS and 1 mM thymidine (Sigma-Aldrich, St. Louis, USA) was added to each well, and the width of the scratches was measured and imaged at 0 h and 48 h. The cell invasion assays were performed in Matrigel-coated 8-µm pore membranes in 24-well transwell chambers (BD Biosciences, New Jersey, USA). A total of 1 × 10⁵ cells were suspended in serum-free medium, seeded into the upper chamber and allowed to migrate toward DMEM containing 10% FBS in the lower side of the chamber for 24~48 h. The migrated cells were fixed in formaldehyde and stained with crystal violet solution for 10 min. The cells that migrated to the lower surface of the insert membrane were counted as invaded cells under a microscope. The chemotaxis assay was similar to the invasion assay, except that the complete medium in the lower compartment was replaced with DMEM containing recombinant human CXCL12 protein ( R&D Systems, USA) as a chemoattractant.