| Products/Services Used | Details | Operation |
|---|---|---|
| Peptide Synthesis | To form the scaffolds, we first oxidized the alginate (Mw ~250 kDa, high G blocks; Novamatrix UP MVG, FMC Biopolymer, Rockland, Maine) with sodium periodate (1.5 %), overnight at room temperature, then quenched the reaction by dropwise addition of ethylene glycol (Sigma) for 45 min. We then dialyzed the solution (MWCO 3.5 kDa) against deionized water for 3 d followed by lyophilization. Afterward, the alginate was dissolved in MES (150 mM MES, 250 mM NaCl, pH 6.5) and covalently conjugated to RGD peptide (GGGGRGDY; GenScript USA Inc., Piscataway, NJ) using carbodiimide chemistry (NHS/EDC). The reaction was continued for 24 h followed by dialysis (MWCO 20 kDa) and lyophilization. This alginate-RGD complex in PBS was then cross-linked via calcium sulfate solution. The gels were casted in desired 24- or 96-well plates followed by two overnight washes to get rid of extra calcium ions and then used as 2D matrices. For 3D structures these same scaffolds were frozen at - 80 °C, lyophilized for 3 d, and stored at 4 °C before cellular studies. | Get A Quote |
T cells recognize mechanical forces through a variety of cellular pathways, including mechanical triggering of the T-cell receptor (TCR) and mechanical triggering of the integrin LFA-1. We show here that T cells can recognize forces arising from the rigidity of the microenvironment. We fabricated 3D hydrogels with mechanical stiffness tuned to 4 kPa and 40 kPa and specially engineered be microporous independent of stiffness. We cultured T cells and antigen presenting cells within the matrices and studied activation by flow cytometry and live imaging. We found there was an augmentation of T-cell activation in the context of mechanically stiffer 3D material as compared to the softer material. In contrast, prolife... More
