For Ca 2+ imaging, as described previously 65 , Jurkat T cells were loaded with 1 μM Fura-2-AM in RPMI 1640 with 10% FCS at room temperature for 25 min. Then cells were washed with centrifugation, resuspended in 1 mM Ca 2+ Ringer's solution, and seeded on poly(acrylamide) (PAAm) hydrogel substrate if not otherwise mentioned. Afterwards, Ca 2+ imaging is acquired immediately. Fluorescence was emitted by 340 nm or 380 nm and infrared images were taken every 5 sec for 25 min at room temperature. The captured images were analyzed by T.I.L.L. Vision software. The traces were analyzed with the software Igor Pro6. For the experiment with BTP-2, all solutions contain 10 μM BTP-2 or the vehicle. The response time is defined as the time period between cell docking on the substrate and Ca 2+ influx. The cells showing elevated Ca 2+ levels ([Ca 2+ ]int exceeding the threshold Ratio 340/380=0.3) were defined as responsive cells. Time 0 of T cell activation is defined as the time point immediately before the [Ca 2+ ]int exceeds the threshold (Ratio 340/380=0.3). The peak was defined as the maximum value of ratio 340nm/380nm within the first 60 sec after the T cell activation. The plateau levels the average of 340nm/380nm ratios from the last 30 sec.