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The ability of activated CD4⁺ T cells to migrate toward CXCL11 was monitored by a chemotaxis assay using Transwell inserts (6.5 mm insert, 3 μm filter, Costar). 600 μl/well RPMI or CXCL11 (100 ng/ml, Recombinant Human CXCL11 (ITAC), carrier-free, Biolegend) was added to the lower chambers. Activated CD4⁺ T cells were resuspended in fresh medium (1 million/ml) and 100 μl was added to the upper chamber of the Transwell inserts, and incubated at 37°C for 3.5 hrs. To evaluate chemotaxis, cells in the bottom chamber were then analyzed by flow cytometry for 1 min to evaluate the number of cells present. Dunn chamber chemotaxis was performed with CD3/CD46-activated T cells allowed to migrate in a gradient of 0-100nM nM CXCL11. Cells were monitored in a temperature-controlled chamber for 30 min by time-lapse imaging using an inverted RMDIB microscope (Leica) equipped with an Orca camera (Hamamatsu) that was controlled by Micromanager image acquisition software (Fiji). The paths of individual cells were tracked using the manual tracking plug-in into ImageJ and tracks analyzed using the chemotaxis tool plug-in into ImageJ (Ibidi).

The impact of inflammatory factors on WJ-MSC migration was analyzed with μSlide plates for chemotaxis evaluation (Ibidi, Gräfelfing, Germany).
For this purpose, 6 μL of WJ-MSC suspension (cell density—1.6 × 10⁶ cells/mL) was injected into the central chamber and incubated for 3 h at 37 °C to allow the cells to adhere to the plate. Two types of medium were then added to the lateral chambers; i.e., control medium without platelet lysate (left side) and selected stimulants (right side) (Figure 2). As a positive control, DMEM with bFGF at 20 ng/mL (cell attractant) was used. Over a 24-h, cell activity was observed under an AxioObserver Z.1 microscope (Carl Zeiss, Oberkochen, Germany) in transmitted light. Cell locations were recorded every 10 min. During the experiment, 37 °C, 5% CO₂ and 21% O₂ conditions were maintained. The obtained image was analyzed qualitatively using the Zen 2.0 program (Zeiss, Oberkochen, Germany)), while quantitative analysis used ImageJ and Chemotaxis Migration Tools (Ibidi, Gräfelfing, Germany). The results are present as p-value Rayleigh test).