Quantitative data of differential adhesion behaviors of Tregs compared to Tconvs on patterns (Fig. 3c-d ) were obtained and analyzed by counting cells on and off the patterns from multiple bright field and IRM images of cells overlaid with fluorescent images of patterns at 100X objective lens after fixing and washing. Unadhered cells were washed off during this washing step by using syringe pumps at a flow rate of 10 μL/min. As IRM images indicate true interaction of cells with surfaces, cells without proper foot print in IRM was considered as non-reactive and excluded from the analysis.
For observation of protein localization, cells were fixed and permeabilized by using 4% paraformaldehyde and 0.1% Triton X-100 after 30 min incubation on patterns in channels and then stained by using standard immunofluorescence techniques24 (link) by flowing reagents in and washing with PBS through the channels by using syringe pumps at a flow rate of 10 μL/min. A polyclonal antibody to PKC- θ (sc-212; C-terminus, Santa Cruz Biotechnology) was used in 5% BSA, followed by Alexa Fluor 568 donkey anti-rabbit IgG (Invitrogen A10042). Actin clusters were stained with Texas RedR-X phalloidin (Life Technologies T7471) stock solution in 5% BSA.
For observation of protein localization, cells were fixed and permeabilized by using 4% paraformaldehyde and 0.1% Triton X-100 after 30 min incubation on patterns in channels and then stained by using standard immunofluorescence techniques24 (link) by flowing reagents in and washing with PBS through the channels by using syringe pumps at a flow rate of 10 μL/min. A polyclonal antibody to PKC- θ (sc-212; C-terminus, Santa Cruz Biotechnology) was used in 5% BSA, followed by Alexa Fluor 568 donkey anti-rabbit IgG (Invitrogen A10042). Actin clusters were stained with Texas RedR-X phalloidin (Life Technologies T7471) stock solution in 5% BSA.