O) at a x molar excess in an effort to occupy excess biotinbinding web-sites, yielding a coating efficiency similarSaitakis et al.eLife ;e..eLife.ofResearch articleBiophysics and Structural Biology Immunologyto PAgels (`Glass Neutr.’, Figure figure supplement C).PDMS gels were coated overnight with .mgmL of fibronectin (Sigma Aldrich, cat.no.F) in PBS.For cell culture T cells were added on PAgel or glass NB001 medchemexpress coverslips for hr; and HeLaCIITA cells had been cultured to confluence for hr around the fibronectincoated PDMS gels before adding T cells (per plate) for extra hr.Untreated or biotinylated (with all the above kit) fibronectin was also utilised to test nonspecific versus distinct binding on PAgels with all the following antibodies antihuman fibronectin rabbit IgG (Sigma Aldrich, cat.no.F) and antirabbit IgG AlexaFluor (Life Technologies, cat.no.A)(Figure figure supplement B).A theoretical surface density of immobilized molecules was calculated.To perform so, we made three assumptions) The volume with the hydrated gel (swollen gel in culture medium) that we can calculate from its thickness as well as the diameter of your slide employed, is larger than the initial volume with the polymerization mix (this PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/21493362 was determined by a prior study [Hynd et al]);) Each of the streptavidinacrylamide molecules in the polymerization mix did polymerize in the gel;) Biotinylated Abs and ICAMFc molecules can only access the initial nm of your gels ( nm being the approximate size on the streptavidin molecule) due to the size on the pore reported in the literature ( nm for PAgels of .kPa and smaller for the extra rigid gels [Trappmann et al ]).In accordance with these assumptions, we calculated the density of immobilized molecules.For the PAgels utilised in cell cultures, mL on the polymerization mix was added in involving two glass coverslips, a single silanefunctionalized ( mm diameter) and one nonfunctionalized ( mm diameter).The gel formed amongst the glass coverslips but in addition formed on the extended surface of your larger mm diameter coverslip.From the measured (by microscopy) gel thickness with the rehydrated PAgels ( mm) plus the diameter of your slide ( mm), we calculated the volume of the hydrated gels .mL.We assumed that the hydrated PAgels possess a bigger volume than their initial polymerization mix (Hynd et al), thus the initial volume of the polymerization mix amongst the coverslips was ranging from to mL.Assuming that each of the streptavidinacrylamide in the initial polymerization mix polymerized, we calculated the quantity of molecules of streptavidinacrylamide in this initial volume.To ascertain the surface density of streptavidinacrylamide, we assumed that biotinylated Abs and ICAMFc molecules could only access the streptavidin inside the 1st nm on the gels, which corresponds for the approximate diameter of a streptavidin molecule (Neish et al) and also, all these molecules could be readily available for binding with soluble biotinylated antibodies taking into account the gel pore size (nm).We thus calculated the powerful surface density of streptavidinacrylamide inside a array of to moleculesmm.Considering that in a lot of the experiments a molar ratio of antiCDICAMFc to antiCD was utilised, the theoretical density of antiCD is of to moleculesmm for to antiCDICAMFc moleculesmm for the .kPa gels.Finally, because the overall protein coating of PAgels, measured by microscopy, was similar around the gels of diverse rigidities (Figure figure supplement C), this array of values for surface density applies to all PAgels applied within the study.