Octadecyl rhodamine b chloride r18

scEMC10 recombinant protein was labeled with FITC using a FITC conjugation kit (Sangon Biotech D601049). Cells were grown on glass coverslips for transfection or treatment as indicated, then treated with FITC-scEMC10 for 2 h, and recovered for the times indicated. The cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. Samples were rinsed three times with PBS (5 min for each wash). Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) for 10 min. Cell membranes were counterstained with Octadecyl Rhodamine B Chloride (R18) (Sigma 83685). Coverslips were rinsed twice (3 min for each wash) with PBS and mounted onto slides using ProLong Gold Antifade reagent (Invitrogen). All images were obtained with the Leica TCS SP8 fluorescence microscope.

Fibronectin and octadecyl rhodamine B chloride (R18) were obtained from Sigma-Aldrich (St. Louis, MO, United States) and Invitrogen (Carlsbad, CA, United States), respectively. IL-1β, IL-6, and TNF-α were obtained from Sino Biological (Beijing, China). Adhesive substrates were prepared from perfusion of microfluidic channels in the BioFlux plate with different concentration of FN at 5 dyn/cm² for 15 min, followed by incubation at RT for 1 h, and blocked with 0.5% BSA at 5 dyn/cm² for 15 min. All cytokine-containing adhesive substrates were prepared from perfusion of cytokines together with FN. MSCs were trypsinized, stained with 300 nM R18, resuspended at 1 × 10⁶ cells/ml in PBS, and perfused from the inlet wells for 10 min at 10 dyn/cm². In experiments that assess the effect of soluble cytokines, cells were incubated with different cytokines for 30 min at RT before perfusion. Images of attached cells after the perfusion were captured at a magnification of 100× using a Nikon TS100 microscope (Nikon Instruments, Inc., Melville, NY, United States) equipped with a CCD camera (QICAM, QImaging, Surrey, British Columbia) and the BioFlux 200 software. Cell number from each image was counted and averaged for each group.

Polymyxin B (PMB; lot number 20120204), amikacin (lot number 058K0803), aztreonam (batch number MKCH8931), and doripenem (lot number 0137Y01) were purchased from Sigma-Aldrich and their solutions were prepared in sterilized Milli-Q water (Millipore, Australia). Phytantriol (98%, 3,7,11,15-tetramethylhexadecane-1,2,3-triol), poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer (Pluronic F127), lipid A (diphosphoryl from Escherichia coli F583), Octadecyl Rhodamine B Chloride (R18), Dulbecco’s modified Eagle’s medium (DMEM, 4.5 g/L glucose), fetal bovine serum (FBS) and [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (tetrazole)] (MTT) were purchased from Sigma-Aldrich; 1,2-dipalmitoyl-d₆₂-sn-glycero-3-phosphocholine (d-DPPC) was purchased in powder form from Avanti Polar Lipids Inc. CellBrite^TM Fix 488 membrane stain was purchased from Biotium. Milli-Q water 18.2 MΩ ∙ cm and D₂O (99.99 atom%) were used in all experiments for preparation of buffers and solutions. All chemicals were used as received without further purification. The Human embryonic kidney 293T cells (ATCC HEK-293T) was the gift from Dr Thomas Naderer, Monash University.