Cells were plated in black wall 96 well plates (1 × 104 cells per well) using a Multidrop 384 dispensing system (Labsystems, Waltham, MA, USA), and experiments were performed the subsequent day. Cells were incubated with various dilutions of CXCL12-GL or GL supernatants as described in figure legends. For competition experiments, cells were incubated with CXCL12 (R&D Systems); CXCR4 antagonist AMD3100 (Sigma, St. Louis, MO, USA); or CXCR7 antagonists CCX733 or CCX754 (gifts of ChemoCentryx) added 30 minutes before incubating cells with CXCL12-GL for 2 hours at 37° C. Cells then were washed twice with PBS before measuring cell-associated bioluminescence. 1 µg/ml coelenterazine in PBS was added to intact cells, and bioluminescence was measured immediately after adding substrate as described above.
Protocol detail
Bioluminescent Assay of CXCL12 Binding
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Other organizations : University of Michigan–Ann Arbor
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Variable analysis
independent variables
- Dilutions of CXCL12-GL or GL supernatants
- CXCL12 (R&D Systems)
- CXCR4 antagonist AMD3100 (Sigma, St. Louis, MO, USA)
- CXCR7 antagonists CCX733 or CCX754 (gifts of ChemoCentryx)
- Cell-associated bioluminescence
- Cells were plated in black wall 96 well plates (1 × 10^4 cells per well) using a Multidrop 384 dispensing system (Labsystems, Waltham, MA, USA)
- Cells were incubated with various dilutions of CXCL12-GL or GL supernatants as described in figure legends
- Cells were incubated with CXCL12, CXCR4 antagonist AMD3100, or CXCR7 antagonists CCX733 or CCX754 added 30 minutes before incubating cells with CXCL12-GL for 2 hours at 37° C
- Cells were washed twice with PBS before measuring cell-associated bioluminescence
- 1 µg/ml coelenterazine in PBS was added to intact cells, and bioluminescence was measured immediately after adding substrate
- Positive control: CXCL12 (R&D Systems)
- Negative controls: CXCR4 antagonist AMD3100, CXCR7 antagonists CCX733 or CCX754
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