Real-time deformability cytometry (RT-DC)

The RT-DC measurements were performed by using the same setup and device described in a previous publication.⁴⁹ Briefly, the cells were trypsinised and were centrifuged at 100g for 5 min. The supernatant was removed, and the cells were resuspended at a concentration of around 5 × 10⁶ cells per ml in 1× PBS buffer with 0.5% (w/v) methylcellulose having viscosity of 15 mPa s. The cell samples were passed through RT-DC devices with 30 μm square channel geometry at a flow rate of 0.16 μL s⁻¹. All the samples were processed at room temperature within 30 min of preparation. All analysis was done using the open source software ShapeOut.⁵⁰ The Young's moduli of the cells were calculated using ShapeOut after filtering out the events falling out of area range of 80–750 μm² and area ratio range of 1–1.05.