Alexa fluor succinimidyl esters

Anti-collagen-1 rabbit pAb (AbCam, Cambridge, UK) was prelabeled with a fluorescent tag and a biotin group to ensure both its fluorescent visibility in nanopatterns and cross-linking to the streptavidin-functionalized PAA gels, respectively. Briefly, 20μL of 1mg/mL antibody sample was incubated for 1 hour with 5μL of ((+)-biotin N-hydroxysuccinimide ester, Sigma-Aldrich; as per the commercial protocol) and 5μL of fluorescent tag kit (Alexa Fluor^® succinimidyl esters, Invitrogen, Molecular Probes ; as per the commercial protocol). Labeled protein then was dialysed overnight in Slide-A-Lyzer MINI Dialysis Device, 7K MWCO (Thermo Fisher) overnight at 4° C in cold PBS, then stored at 4° C in the darkness. 10μL droplets of 0.1mg/mL labeled antibody solution were then placed atop of the 5×5mm or 1×1cm square micro- or nano-stamps. To ensure a proper coverage and effective stamp surface coating with labeled α-collagen-1 antibody, the antibody solution droplet was ‘‘sandwiched’’ between the stamp’s printing surface and 15mm round glass coverslip (Carolina, USA), which had been baked in the furnace for 5–10 hours at 450° C.

The markers for T-cell activation and proliferation were analyzed by a FACScanto II (BD Biosciences, Heidelberg, Germany) flow cytometer. Antibodies used in this study were: PerCP-labeled anti-human CD4 (clone RPA-T4), PE-Cy7 labeled anti-human CD45RA (clone HI100), Alexa Fluor 700-labeled anti-CD27 (clone O323), FITC-anti-human CD25 (BC96), all purchased from (BioLegend, San Diego, CA, USA). Alexa Fluor 647-labeled anti-human CD11a (LFA-1: TS1/22.1.1.13) was prepared from hybridoma cells (ATCC No. HB 202) and conjugated using Alexa Fluor succinimidyl esters (Invitrogen, Carlsbad, CA, USA). Dead cells were stained with a LIVE/DEAD Fixable Dead Cell Stain Kit (L34957, Invitrogen, Carlsbad, CA, USA) and were gated out during analysis. For cell sorting, a FACSaria II cytometer was used. The data were analyzed by FACSdiva software and reanalyzed by Flowjo ver 8.8.7 (Tree Star Inc., CA, USA).