Fluorescein isothiocyanate (fitc)

The 1 mg GPC1 antigen was dissolved in 0.1 M carbonate buffer of 0.5 mL pH9.0. FITC (sigma F4274) was dissolved with DMSO, and the final concentration of FITC was 1 mg/mL. FITC-DMSO solution was slowly added to antigen solution, and 50 μl FITC-DMSO solution was added to antigen solution. The reaction time was 8 h at 4°C. The reaction was terminated by adding NH₄CL with the final concentration of 50 mM and reacted at 4°C for 2 h. Ultrafiltration removes unreacted FITC.
The isolated rabbit PBMC was suspended to the final volume of 20 μl PBS and 2 μl GPC1-FITC was added. The PBMC was gently vortexed and blended. The light-avoiding reaction lasted 1 hour at room temperature (a small number of cells without GPC1-FITC were reserved as negative control). After the reaction, 1 mL PBS was added; the supernatant was discarded by centrifugation for 5 minutes and precipitated by 1 mL PBS. The supernatant was discarded by centrifugation for 5 minutes and precipitated by 1 mL PBS. The samples were prepared by centrifugation for 5 min at 400 ×g, discarding the supernatant, and resuspension precipitation with 500 μl PBS. The density of the samples was about 10⁷/mL.

To label the phage capsid with amine-reactive FITC, the buffer of purified phages (1 × 10¹¹ p.f.u.) was changed using Amicon Ultra-15 centrifugal filter devices (Millipore) to 0.1 M carbonate buffer pH 9.0, and FITC (Sigma-Aldrich) was added to a final concentration of 0.25 mg ml⁻¹. After incubation for 1 h at room temperature with rotation in the dark, unbound dye was removed via buffer exchange into 25 mM sodium phosphate, pH 7.4, 150 mM NaCl using centrifugal filter devices. To label with amine-reactive DSB-X biotin, the DSB-X Biotin Protein Labeling Kit (Molecular Probes) was used according to the manufacturer’s instructions. Briefly, phages (4 × 10¹⁰ p.f.u.) were resuspended in 220 µl of 0.1 M carbonate buffer, pH 8.5, and DSB-X biotin was added to a final concentration of 0.09 mg ml⁻¹. After incubation for 1.5 h at room temperature with stirring, excess biotin was removed by purification resin.

FITC-labeled S.pn were prepared by incubation with 4 mg/ml FITC (Sigma, Poole, UK) for 30 min at 4°C. Peritoneal neutrophils (5 ×10⁵ cells) were incubated with FITC-labeled bacteria (at multiplicity of infection, MOI, of 100) for 30 min at 37°C. After washing steps, cell nuclei were stained with DAPI (Invitrogen), followed by visualization using confocal laser scanning microscopy (LSM 510, Zeiss). The ratio of engulfed bacteria (as determined by overlay of green bacteria) was quantified by an independent researcher from 100 counted cells per well and was expressed as percentage of cells that contain bacteria.

P. gingivalis was labeled with fluorescein isothiocyanate (FITC) by incubating washed bacteria with 0.1 mg/ml FITC (Sigma-Aldrich) in carbonate buffer (pH 9.5) for 20 min at room-temperature (RT). 4x10⁶ shCtrl and shVCL THP1 cells were differentiated in 10 cm² petri dishes and infected with FITC-labeled P. gingivalis at MOI 100 for 1.5 h. Cells were extensively washed and then collected using 5mM ethylenediaminetetraacetic acid (EDTA) (pH 7.5) at 4°C for 1 h, followed by fixation with 4% PFA in fluorescence-activated cell sorting (FACS) buffer containing 5mM EDTA. FITC-positive cells were analyzed by using a BD Accuri C6 Plus Flow Cytometer.

CHS was induced using the hapten FITC (Sigma–Aldrich) as described previously with minor modification [59 (link)]. In brief, 100 μl 0.5% FITC (Sigma–Aldrich) resuspended in acetone and DBP (Sigma–Aldrich) (at a ratio of 1:1) was painted onto the shaved skin of WT and p38α^ΔDC mice on Days 0 and 1. On Day 6, the baseline ear thickness was measured, and the mice were challenged by applying 20 μl of 0.5% FITC or vehicle onto the contralateral ear. Ear thickness was measured, and the mice were sacrificed for analysis 24 h later. Skin cells were prepared as previously described [63 ].