Celltracker red cmptx dye

In addition, we observed the above phenomena using confocal microscopy (TiEA1R, NIKON INSTECH Co. Ltd., Tokyo Japan) under the conditions of with or without AIM coating. Debris was stained with FVD520 (eBioscience). The peritoneal cells derived from the zymosan model mice were stained with APC anti-mouse F4/80 antibody (BioLegend, San Diego, CA) and incubated with Anti-APC microbeads (Miltenyi Biotec). F4/80 positive cells were separated using a MACS LS column (Miltenyi Biotec) stained with CellTracker™ Red CMPTX Dye (10 µM) (Thermo Fisher Scientific).

For migration assays, Corning^® FluoroBlok™ Cell Culture Inserts with a pore size of 8 μm combined with 24‐well cell culture plates were used. Before start of the migration assay, the cells were serum‐starved overnight in RPMI with 1% BSA. Cells were then stained with CellTracker™ Red CMPTX dye (Thermo Fisher) at a final concentration of 10 µM for 30 minutes in serum‐free RPMI without phenol red. Cells were pelleted and again incubated in serum‐free RPMI for 30 minutes without phenol red to remove the surplus dye. Cells were seeded in duplets onto the insert membrane at a density of 3 × 10⁵ cells/300 µL per insert. The bottom compartment was filled with 1.0 mL of the same medium. After 30 minutes rest period, cells were placed into wells containing the specified chemoattractant and were allowed to migrate through the membrane for 48 hours. Migration was quantified from the bottom of the wells by fluorescence measurement using the CLARIOstar microplate reader (BMG; Extinction: 577 nm; Emission: 607 nm) every 10 minutes within the first two hours and then every 30 minutes for 46 hours. Cell migration was displayed as signal compared to the measurement at 4 hours.

S. aureus (ATCC 6538, Sigma Aldrich, Saint Louis, MO, USA) and S. epidermidis (ATCC 12228, Sigma Aldrich) were plated on agar plates (Columbia agar with 5% sheep blood, BD) and incubated at 37 °C overnight. Single colonies were scraped and suspended in 2 mL sterilized rich medium (Bacto Tryptone 15g/L, BD) for 2 h. Bacterial suspensions were then stained using a CMPTX red dye (Cell Tracker red CMPTX dye, ThermoFisher, Waltham, MA, USA). Specifically, a 10 mM stock solution of the dye was prepared diluting the lyophilized product with dimethyl sulfoxide (DMSO, HPLC grade, 99.9+%, Thermoscientific, Waltham, MA, USA). The stock solution was then diluted to a working concentration of 2 mM in the final bacterial suspension volume. The suspension was then vortexed and incubated for 30 min at 37 °C. After incubation, the suspension was centrifuged at 4000 rpm for 15 min to recover the stained bacteria. Finally, the supernatant was discarded and the bacterial cells were suspended in RPMI medium without antibiotics.