Page 59 - Live-cellanalysis handbook

Kinetic Chemotaxis Assays

A B

Figure 7. Evaluation of ClearView chemotaxis assay gradient. (A) 10,000 kDa dextran, labeled with Alexa Fluor® 594, was added to the reservoir plates for
the ClearView and 96-well modified Boyden-chamber at a concentration of 10 μM to establish gradients over 72, 48, 24 and t=0 hours. Measurements of
diffusion were made by sampling the insert wells on both plates and measuring fluorescent intensity on a microplate reader. Each data point represents
mean ± SEM, N=3. (B). An FBS gradient was established over 72, 48, 24 and t=0 hours in separate wells. Serum starved NucLight Red HT-1080 cells
were added at a density of 500 cells per insert well to the established gradients and FBS-induced chemotactic response was measured. Each data point
represents mean ± SEM, N=3.

Conclusions References

The IncuCyte chemotaxis assay is a quantitative and reproducible 1. Taylor, L., Brodermann, M., McCaffary, D., Iqbal, A. J., and Greaves, D. R:
approach for measuring chemotaxis in both adherent and non- Netrin-1 reduces monocyte and macrophage chemotaxis towards
adherent cell types. This assay format allows for the kinetic complement component C5a. PLOS PLoS ONE (2016) 11(8): e0160685.
detection of cell migration toward chemotactic gradients on 2. Kawaguchi A, Orba Y et al: Inhibition of the SDF-1a-CXCR4 axis
a physiologically relevant substrate, with movies and images by the CXCR4 antagonist AMD3100 suppreses the migration of
that support quantitative measurements and provide associated cultured cells from ATL patients and murine lymphoblastoid cells
morphological and phenotypic insights. from HTLV-U Tax transgenic mice. Blood 2009, 114: 2961-2968.

Key features demonstrated in the IncuCyte chemotaxis assay are:

• Kinetic cell migration is automatically processed on both the
top and bottom side of the optically clear membrane, allowing
for real-time analysis of chemotactic driven migration of
adherent and non-adherent cell types.
• Non-adherent cell migration is precisely quantified in a
physiologically relevant environment, while the plate stays
stationary, allowing for the evaluation of rare primary
hematopoietic cells from the blood.
• Cells are required to migrate across a biologically relevant
surface, demonstrating the need for cell-surface interaction,
providing an opportunity for evaluation of integrin receptor
signaling.
• All data points can be validated by individual images or time-
lapse movies to confirm processing metrics, significantly
enhancing the confidence in the measured response.

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