Metamorph imaging software

ROS formation was assayed using dihydrorhodamine 123 (DHR) as described by Palomba et al. [35 (link)]. Briefly, AGS cells infected with H. pylori, loaded with DHR (10 µM for 20 min), were treated for 1 h with (1) Hp phage (Hp φ; 10⁶ PFU/mL); (2) lactoferrin adsorbed on hydroxyapatite nanoparticles (LF-HA; 200–600 µg/mL); (3) the complex Hp φ +LF-HA described above, and analysed with a Leica DMI6000 fluorescence microscope equipped with a Leica DFC320 cooled digital CCD camera (Leica Microsystems). The excitation and emission wavelengths were 488 and 515 nm, respectively. Images were collected with exposure times of 100–400 ms, digitally acquired, and processed for fluorescence determination at the single cell level with Metamorph Imaging Software (Leica MetaMorph © AF, Wetzlar, Germany). Mean fluorescence values were determined by averaging the fluorescence values of at least 50 cells/treatment.

Dissected fetal livers were fixed in 4% Paraformaldehyde (PFA) overnight, before they were soaked in 30% sucrose and mounted in OCT compound. 10μm sections were prepared using a cryostat. The sections were incubated in blocking buffer (PBS with 10% FBS, 0.05% Tween20 and 10% goat serum (DAKO)) for 1 hour before the sections were incubated with primary antibodies at 4°C overnight in blocking buffer.
Primary antibodies used in this study were rabbit anti-GFP (598, polyclonal, MBL) (1/200); and purified rat anti-mouse CD31 (553370, MEC13.3, BD Biosciences) (1/100).
Sections were washed three times in PBST (PBS with 0.05% Tween20) for 15 minutes each and then incubated with fluorochrome-conjugated secondary antibody at room temperature for 1 hour.
Secondary antibodies used in this study include Alexa Fluor 488 Goat Anti-Rat IgG (A11006, Life Technologies); and Alexa Fluor 647 F(ab')₂ Fragment of Goat Anti-Rabbit IgG (H+L) (A21246, Life Technologies). All secondary antibodies were used at 1/400 dilution.
Sections were further washed three times in PBS and mounted using Prolong Gold anti-fade medium with DAPI (Life Technologies). Images (of Alexa Fluor 488, Alexa Fluor 647, DAPI and endogenous RFP) were taken using a low-light time lapse microscope (Leica) using the Metamorph imaging software and processed using ImageJ.

ROS formation was assayed using dihydrorhodamine 123 (DHR) as described by Palomba et al. (21 (link)). Briefly, using the co-culture system, microglial cells were incubated with 10 μM DHR (20 min) and the differentiated Caco-2 cells were plated on the transwell inserts and treated as detailed above. Finally, microglial cells were analyzed with a Leica DMI6000 fluorescence microscope equipped with a Leica DFC320 cooled digital CCD camera (Leica Microsystems). The excitation and emission wavelengths were 488 and 515 nm, respectively. Images were collected with exposure times of 100–400 ms, digitally acquired and processed for fluorescence determination at the single cell level with Metamorph Imaging Software (Leica MetaMorph © AF). Mean fluorescence values were determined by averaging the fluorescence values of at least 50 cells/treatment condition/experiment.

ROS formation was assayed using dihydrorhodamine 123 (DHR) as described by Palomba et al. [19 (link)]. Briefly, primary cortical neurons, loaded with DHR (10 µM for 20 min), were processed as detailed in the figure legends and analyzed with a Leica DMI6000 fluorescence microscope equipped with a Leica DFC320 cooled digital CCD camera (Leica Microsystems, Milan, Italy). The excitation and emission wavelengths were 488 and 515 nm, respectively. Pictures were collected with exposure times of 100–400 ms, digitally acquired and analyzed for fluorescence determination at the single cell level with the Metamorph Imaging Software (Leica MetaMorph© AF). Mean fluorescence values were determined by averaging the fluorescence values of at least 50 cells/treatment condition/experiment.