Migration Assays
Immune cells play an important regulatory role in immune homeostasis. To support this process, migration of immune cells is critical for the delivery of protective immune responses to tissues.
Chemotaxis
Neutrophil migration through the HUVEC monolayer
Treatment with TNFa significanty increased fMLP-induced neutrophil migration through HUVEC monolayers (ns=not significant; **p<0.01; ***p<0.001±SEM). fMLP is a chemoattractant.
The figure shows test antibody inhibits CCL2 induced THP-1 (monocytic cell line) at higher treatment concentrations (n=2; ± SEM). Isotype treatment has no effect on cell migration.
THP-1 migrated cell counts for the 4 control conditions: untreated, VEGF-165 treated, Axitinib and PI3K Inhibitor.
THP-1 migrated cell counts are indicative of the number of THP-1 cells which have passed through the HUVEC monolayer into receiver wells as a direct measure of the permeability of the HUVEC monolayer following drug treatment. Axitinib/VEGF-165 and PI3Kinhibitor/VEGF-165 co-treatment demonstrates significant inhibition of MCP-1 induced transmigration of THP-1 cells suggesting inhibition of HUVEC monolayer permeability. Each condition is representative of three technical replicates (n=3); mean ± SEM.
The figure shows the test antibody inhibits primary CD4+T cells (anti-CD3/anti-CD28 activated) migration towards CCL20 (n=3; ± SEM) in a dose dependent manner. Cell migration was analysed using Cell-Titre Glo and plotted as relative luminescence units (RLU).
THP-1 migration induced by MCP-1/CCL2
THP-1 cells were stained with Calcein AM and added to the top chamber of the transwell. MCP-1 was then added to the bottom chamber in the presence or absence of Wortmannin (PI3K inhibitor). At the end of the 2 hour migration, the number of cells that migrated into the basal chamber of the experimental set-up was quantified using the JuLITM Stage imaging platform. Results were graphically presented using GraphPad Prism (Version 8.0.2) software. Relative fold-changes were also determined for each treatment condition using the negative (untreated) condition as a baseline. Wortmannin serves as a positive control for the inhibition of THP-1 migration.
Each condition is representative of three independent biological repeats (**p<0.05, n=3±SEM). MCP-1 induced the migration of THP-1 cells, having an estimated and significant two-fold-change increase relative to the untreated condition (negative control), suggesting its suitability as a chemoattractant for these cells. Treatment of THP-1 cells with the PI3K enzyme inhibitor Wortmannin resulted in a significant reduction in cell migration when compared to the MCP-1 only condition.
Primary monocyte migration induced by SDF-1
Human peripheral blood derived CD14+ monocytes were stained with Calcein AM and added to the top chamber of the transwell. SDF-1was then added to the bottom chamber in the presence or absence of CXCR4 inhibitor. At the end of the 4 hour migration, the number of cells that migrated into the basal chamber of the experimental set-up was quantified using the JuLITM Stage imaging platform. Results were graphically presented using GraphPad Prism (Version 8.0.2) software. Relative fold-changes were also determined for each treatment condition using the Vehicle control.
Each condition is representative of three independent biological repeats (**p<0.05, n=3±SEM). SDF-1 induced the migration of CD14+monocytes suggesting its suitability as a chemoattractant for these cells. Treatment of CD14+monocytes with CXCR4 inhibitor resulted in a significant reduction in SDF-1 induced cell migration.
Peripheral blood neutrophils migration induced by IL8
Human peripheral blood derived neutrophils were stained with Calcein AM and added to the top chamber of the transwell. Recombinant IL8 was then added to the bottom chamber. At the end of the 90 minutes migration, the number of cells that migrated into the basal chamber of the experimental set-up was quantified using the imaging platform. Results were graphically presented using GraphPad Prism (Version 8.0.2) software. Each condition is representative of three independent biological repeats (**p<0.05, n=3±SEM).
Chemokinesis
A scratch was formed on a A549 cell monolayer and treated with Olaparib (Ola), 5-Fluorouracil (5-FU), or a combination of both. Images were acquired using a live imaging platform (DETAILS) at the indicated time points (hrs). The wound size/scratch was measured at all time points. The wound closure percentage was analysed assuming 0% closure at 0 hours, and calculating all the following time points as ratio to 0 hours [ *p<0.05; **p<0.01;***p<0.001; ± SEM].
HUVEC transmigration in response to SDF-1 or VEGF-165. To assess HUVEC transmigration in the presence of different chemoattractants, 5×10 HUVEC cells were added to the apical chamber of Boyden-Chambers. The basal chamber contained 50 ng/ml VEGF-165, 100 ng/ml SDF-1 or serum free media (control). Cells were left to migrate for 4 hours at 37C, 5% CO2. After 4 hours, non-migrated cells in the apical chamber were removed with a cotton swab. Migrated cells attached to the underside of the apical chamber were stained with 1µM Calcein-AM (30mins) and imaged using JuLl-Stage. A) Fold change in HUVEC migration normalized to the control B) Representative images for each condition. C) Schematic representation of experimental procedures.
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