Chemotaxis assays were performed as described previously 53 (link). 5×105 enriched CD4 splenic T cells were incubated for 1 h with 1× DMEM containing 0.5% fatty acid–free BSA (EMD Biosciences), 5% antibiotics, L-glutamine (Cellgro), and Hepes. Cells were then allowed to migrate through 5-μm-pore–sized transwells (Corning) toward soluble CXCL12, CXLC13 (R&D Systems) or media alone, for 3 h at 37°C. Cells were collected, stained, and resuspended in 45 μl of staining buffer and analyzed by flow cytometry.
Protocol detail
Chemotaxis Assay for CD4+ T Cells
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Antibiotics
Assays
Buffer
Cd4 cells
Cells
Chemotaxis
Cxcl12
Fatty acid
Flow cytometry
Hepes
L glutamine
Splenic
Corresponding Organization :
Other organizations : Yale University
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Variable analysis
independent variables
- Chemotactic stimuli (CXCL12, CXCL13, or media alone)
- Cell migration through transwells
- Enriched CD4 splenic T cells (5×10^5 cells)
- Incubation time (1 h)
- Culture medium (1× DMEM containing 0.5% fatty acid–free BSA, 5% antibiotics, L-glutamine, and Hepes)
- Transwell pore size (5 μm)
- Incubation time for migration (3 h)
- Temperature (37°C)
- Positive control: CXCL12 and CXCL13 (chemotactic stimuli)
- Negative control: Media alone
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