Atto 565 biotin

Atto565, Atto565-biotin, Atto565-NHS and all the amino acids used were obtained from Sigma-Aldrich (St. Louis, MO). Bacterial strain BW25113 was obtained from Yale E. coli Genetic Stock Centre, BL21(DE3) strain was obtained from New England Biolabs (Ipswich, MA) and MG1655WT, ΔOmpF, ΔOmpC and ΔOmpF&C were kindly provided as a gift by Dr. Chang-Ro Lee (Department of Biological Sciences and Bioinformatics, Myongji University, Yongin, South Korea).

The streptavidin-coated Spherotech SVMH-400-4 superparamagnetic beads (diameter: 32 μm) were coupled with Atto 565-biotin (Sigma-Aldrich) and biotinylated poly-L-lysine molecules through the streptavidin-biotin reaction. We placed 5 μL streptavidin-coated magnetic bead solution into an Eppendorf tube. The bead solution was washed three times with phosphate-buffered saline (PBS) to remove preservatives. A permanent magnet was placed under the tube to collect the magnetic beads. The beads were then collected by a micropipette and resuspended in 90 μL of Milli-Q. Atto 565-biotin (1 mg) was diluted in 200 μL of ethanol. Five μL of this dilution and 5 μL of the biotinylated adhesion molecule solution (1 : 1000 in PBS) were mixed with 90 μL of the magnetic bead suspension for 30 minutes. Finally, the solution was washed with PBS five times to remove the biotin surplus through magnetic separation, and the supernatant was collected using a micropipette.
In microinjection, a microneedle pulled from a glass capillary tube using a micropipette laser puller was loaded with mineral oil. One magnetic bead each time was aspirated into the microneedle using a microinjector (CellTram 4r Oil, Eppendorf) and then injected into the center of the middle and distal regions of the mouse mandibular arch.