Dynamic Mass Redistribution (DMR) as a Method to Enable Real-time Investigation of CKR Signalling in Live Human T Cells


K BarkanG OsborneK BennettM LeveridgeN DavisC Fieldhouse
1 Sosei Heptares , UK


Objective. Interaction of chemokines with conventional chemokine receptors (cCKR) promote specific trafficking of leukocytes. Here, we describe the use of label-free dynamic mass redistribution (DMR) to enable real-time investigation of cCKR responses in human T cells. This method allows increased throughput and reproducibility when compared to conventional chemotaxis assays.

Methods. T cells were isolated from human PBMCs (EasySep™ Human T Cell Isolation Kit), activated (Immunocult™ CD3/CD28/CD2) and cultured in IL-2 supplemented medium. Cell-surface expression of T cell markers and 15 cCKR were investigated using flow cytometry over 16 days. T cells (+/- PTX) were seeded, allowed to settle for 2 hrs and stimulation with antagonist or agonist prior to reading on a Corning® Epic® BT System. Following a 1 hr incubation, T cells were stimulated with predetermined EC80 CXCL11 concentration and inhibition of maximum response fitted.

Results. CXCR3, CXCR4, CCR4 and CCR7 were detected on >60% of CD3+ T cells and CCR10, CCR6, CXCR5 and CCR8 on >25% at a given timepoint. CCR1, CCR2, CCR3, CCR5, CCR9, CX3CR1 and CXCR6 were not detected. T cells from multiple donors demonstrated reproducible, concentration dependent, PTX sensitive responses to CXCR3 agonists (CXCL11>CXCL10>CXCL9: pEC50 10.03 ±0.1, 9.26 ±0.1 and 8.30 ±0.2, respectively). Previously described CXCR3 antagonists1 were able to inhibit the CXCL11 stimulated response (pIC50 9.37 ±0.36 and 9.24 ±0.38, respectively).

Conclusion. T cells isolated and activated from PBMCs can be cultured and used to investigate CXCR3, CXCR4, CCR4 and CCR7 signalling in vitro. Flow cytometry identified the correct time-point should be used to achieve a T cell population with the highest cCRK expression. Importantly, DMR offers an alternative tool to determine the potency of CKR antagonists in a native, label free setting; a challenge for traditional chemotaxis.

Hosted By

The European Laboratory Research & Innovation Group Our Vision : To provide outstanding, leading edge knowledge to the life sciences community on an open access basis
Event Logo Find us on Facebook Follow us on Twitter

Get the App

Get this event information on your mobile by
going to the appstore or google play and search for 'elrig'
Eventflo Home
copyright ELRIG,, Labhoo Ltd 2003-2021