Penicillin-streptomycin influences macrophage mechanical properties and microenvironment mechano-sensation.

Penicillin-streptomycin (pen-strep) is routinely included in cell culture media, yet its impact on macrophage mechanics has not been systematically examined. Here, we show that pen-strep treatment increases macrophage stiffness in a time-dependent manner, while adhesion strength is only transiently affected. Morphological analysis revealed that pen-strep promotes cell spreading on PDMS rubber, collagen I, laminin, poly-amino acids, and poly-RGD peptides, but reduces spreading on type IV collagen, indicating altered extracellular matrix sensing in a context-dependent fashion. Gene expression assays further demonstrated upregulation of YAP-1 and TAZ and downregulation of β1 integrin, consistent with reprogramming of mechanotransduction pathways. Consequently, pen-strep elevated intracellular ROS, suppressed the M1 gene spectrum, induced heterogeneous M2-associated responses, and impaired phagocytic capacity. Collectively, these findings identify pen-strep as a modulator of macrophage stiffness, ECM mechano-sensation, polarization, and key immune functions, raising concerns about its routine use in mechanobiology research and clinical applications.