Chemotaxis Assay for CXCR5+ T and B Cells

Chemotaxis assays were carried out as described previously45 (link),51 (link), using 96-well transwell plates with 5 μm pores (Corning). CXCR5⁺ CD8⁺, CD4⁺ T cells and CD19⁺ B lymphocytes as well as CXCR5^- CD3⁺ T cells were sorted from patient PBMCs and rested overnight in complete medium. 2.5 x 10⁴ cells were seeded onto upper wells in 100 μl medium. The bottom wells contained 235 μl PBS with or without 1 μg/ml recombinant human CXCL13 (R&D). After 3 hrs, 50 μl Precision Count Beads™ (Biolegend) were added and transmigrated cells were counted by flow cytometry. Cell numbers were calculated using the following formula: absolute cell count = cell count / bead count x total bead concentration. The chemotactic index was calculated as the ratio of transmigrated CXCR5⁺ cells over transmigrated CXCR5^- control T cells.

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Thommen D.S., Koelzer V.H., Herzig P., Roller A., Trefny M., Dimeloe S., Kiialainen A., Hanhart J., Schill C., Hess C., Savic Prince S., Wiese M., Lardinois D., Ho P.C., Klein C., Karanikas V., Mertz K.D., Schumacher T.N, & Zippelius A. (2018). A transcriptionally and functionally distinct PD-1+ CD8+ T cell pool with predictive potential in non-small cell lung cancer treated with PD-1 blockade. Nature medicine, 24(7), 994-1004.

Publication 2018

96-well transwell plates with 5 μm pores recombinant human CXCL13 Precision Count Beads™

Assays B lymphocytes Cd4 t cells Cells Chemotaxis Cxcl13 Cxcr5 Flow cytometry Human Patient T cells

Corresponding Organization : University Hospital of Basel

Other organizations : Roche (Switzerland), University of Lausanne, Ludwig Cancer Research, Oncode Institute, The Netherlands Cancer Institute

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Variable analysis

independent variables

Presence or absence of 1 μg/ml recombinant human CXCL13 in the bottom wells

dependent variables

Number of transmigrated CXCR5+ CD8+, CD4+ T cells and CD19+ B lymphocytes
Number of transmigrated CXCR5- CD3+ T cells

control variables

Cell type (CXCR5+ CD8+, CD4+ T cells, CD19+ B lymphocytes, CXCR5- CD3+ T cells)
Cell seeding density (2.5 x 10^4 cells per upper well)
Incubation time (3 hours)
Transwell plate with 5 μm pores

controls

Negative control: PBS without CXCL13 in the bottom wells

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