No 1.5 glass coverslip

Cervical cancer HeLa cells were grown in high glucose-Dulbecco’s Modified Eagle Medium (DMEM, Life Technologies) supplemented with 10% cosmic calf serum (Hyclone), 4 mM sodium pyruvate (Life Technologies) and 1% penicillin–streptomycin (Life Technologies). HeLa cells were maintained in cell culture incubator at 37 °C with 5% CO₂. For all imaging experiments, cells were plated onto 25 mm round No.1.5 glass coverslips (Thermo Scientific), in their growth medium and grown to 70–80% confluence. All imaging experiments were done in DMEM Fluorobrite (Life Technologies) without serum.

Microfluidic chips with two inlets and three chambers for cell culture (illustrated in Fig. 1a) were made in PDMS (Sylgard 184, Dow Corning Corporation, MI) and bonded to a No. 1.5 glass coverslip (ThermoScientific, MA). Pipet tips (200 μl) were inserted into 1/16″ tygon tubing connected to steel tubes (16-gauge) into punched inlets and outlets to act as fluid reservoirs.
To prepare for experiments, devices were sterilized with 70% ethanol then abundantly flushed with sterile PBS. Channel surfaces were pacified by flushing the devices with complete culture medium and incubating >12 hrs. Cells were then seeded at appropriate density and allowed to adhere for 12 hrs under no-flow conditions (all reservoirs at equal height). Medium was replaced then and again 24 hrs and 4 hrs before an experiment.
For treatment with a pulse of TNF, medium with TNF at the final desired concentration was prepared with 1 μg/ml Alexa Fluor^® 647 conjugated-BSA (Life Technologies, MA). The device was securely mounted on a custom stage for a BD Pathway 855 BioImager and flow allowed to reach a steady state in the pre-pulse mode (Fig. 1a) while monitoring by imaging the Alexa-647-BSA epifluorescence signal. To pulse cells with TNF, the ‘medium + TNF’ reservoir was temporarily raised manually (Fig. 1a). Pulse duration was verified by imaging the Alexa-647-BSA epifluorescence signal.

To perform q‐DC, standard soft lithography methods were used to fabricate microfluidic channels in PDMS. A mixture of 10:1 ratio of base to cross‐linker (Sylgard 184, Dow Corning) was poured onto a master wafer containing bifurcating channels.^[47] After curing, the PDMS device layer was bonded to a No. 1.5 glass coverslip (Thermo Fisher) using plasma treatment (Plasma Etch, Carson City, NV). Within 48 h of device fabrication, cell suspensions of 1 × 10⁶ cells mL⁻¹ were driven through constrictions of 9 µm (width) × 10 µm (height) by applying 69 kPa of air pressure. The images of cells were captured during deformation through the constrictions using a CMOS camera with a capture rate of 1600 frames s⁻¹ (Vision Research, Wayne, NJ) mounted on an inverted Axiovert microscope (Zeiss, Oberkochen, Germany) equipped with a 20×/0.4NA objective. To analyze the time‐dependent shape changes of individual cells during deformation, a custom MATLAB (MathWorks, Natick, MA) code (https://github.com/rowatlab) was used.^[47] To determine the mechanical stresses applied to individual cells, devices that had been calibrated with agarose particles of defined elastic modulus as previously described were used.^[48] Stress–strain curves were obtained for single cells and a power‐law rheology model was subsequently fitted to the data to yield measurements of elastic modulus, fluidity, and transit time.