Lab tek chamber slide system

Stably transfected esophageal adenocarcinoma cells were seeded into 4-chambered glass slides (Nunc Lab-Tek Chamber Slide System). Cells were then incubated overnight. After 24 hours, cells were rinsed with PBS, fixed with 3.7% w/v paraformaldehyde (Sigma), rinsed with PBS, and permeablized in 0.5% Triton X-100 (Sigma). Nonspecific immunoglobulin binding was blocked with 5% normal goat serum and 0.5% NP-40 (Sigma). Primary antibodies recognizing with STMN-1 (abicam) were diluted 1:100 in blocking solution. After incubation with primary antibody, cells were rinsed with 0.05% Tween-20 (Bio-Rad) in PBS, and then incubated with secondary antibody for 1 h at RT. Stain with 3,3′-Diaminobenzidine (DAB) and observed under light microscope.

R2J-2D cells were set on a Lab-Tek-Chamber Slide system (Nalge Nunc International, Rochester, NY, USA) and were incubated with 5 µM of Fluorescein Diacetate (FDA, Thermofisher Scientific, Courtaboeuf, France), 1µM Hoechst 33342 (Interchim, Montluçon, France), and 10mg/mL Propidium Iodide (PI) (Interchim) in a 5% CO₂ incubator, 37 °C, 20 min. After staining, cells were installed on the microscope incubation chamber with a controlled atmosphere at 37 °C and 5% CO₂ (POC Chamber, Pecom, Erbach, Germany). Images were collected with a Leica TCS SP2 AOBS (Acoustico Optical Beam Splitter) inverted laser scanning confocal microscope equipped with an ×63 oil immersion objective (HCX PL APO 63.0× 1.40). Laser excitation and emission (adjusted with AOBS) were, respectively, 351–364/425–485 nm for Hoechst, 488/500–540 nm for FDA, and 543/600–650 nm for PI. Confocal pinhole (Airy units) was 1 for all channels. Each experiment was performed on a randomly chosen field. Raw image merging was obtained by LCS Lite software (Leica LCS, version 2.61, Overlay).

S. gordonii cells (10⁸) were stained with hexidium iodide and aerobically cultured in CDM containing 0.025% glucose for 16 h using an 8-well saliva-coated LAB-TEK Chamber Slide System (Nalge Nunc International, Naperville, IL). For analysis of mixed biofilm formation, S. gordonii, an early colonizer of the tooth surface, was first cultured the same way as described above, washed gently twice by PBS, and then co-cultured with 1.5 × 10⁷F. nucleatum cells labeled with 5-(and-6)-carboxyfluorescine succinimidyl ester, as described previously (5 (link)). The bacterial cells (1.5 × 10⁸) were then used in an arginine-supplemented mixed biofilm experiment. S. gordonii-P. gingivalis-mixed biofilms were also generated in the same manner. As for the F. nucleatum mono-species biofilm formation, FITC-labeled organisms (2.5 × 10⁷ cells) were cultured in PBS with specific metabolites for 24 h. Biofilms were observed with a Zeiss LSM 510 confocal laser scanning microscope (Carl Zeiss MicroImaging GmbH, Germany). Obtained images were analyzed using the Imaris 7.0.1 software package (BitplaneAG, Zurich, Switzerland), as described previously (38 (link)).

S. xylosus strain C2a expressing cyan fluorescent (C2a-B2) was used [33 (link)]. This strain contains the erythromycin-resistance plasmid pJEBAN2. The strain C2a-B2 was pre-cultivated in Brain Heart Infusion (BHI, Becton, Dickinson and Company, Le Pont de Claix, France) with 10 µg/mL erythromycin at 30 °C, with stirring at 170 rpm for 24 h. Bacterial concentration was then measured by determining the optical density at 600 nm (OD₆₀₀) and appropriate dilution was prepared. The strain was then inoculated at 10⁶ CFU/mL in BHI in the Lab-Tek chamber slide system (1 chamber borosilicate cover glass system, NUNC 15536, 8.6 cm²) and incubated at 30 °C for 9, 24 and 48 h in a humid chamber. After incubation, the supernatant in the Lab-Tek chambers was removed, the adhered cells were washed twice with tryptone salt and then were detached by scratching in tryptone salt. The bacterial population of biofilm after 9, 24 and 48 h were determined by 10-fold serial dilutions on BHI agar plates and enumerated after 24-h culture at 30 °C. In parallel, the cells detached by scratching in tryptone salt were pelleted by centrifugation for 2 min at 4500× g and at 4 °C and the pellet was immediately frozen in liquid nitrogen and stored at −80 °C before extraction of RNA. Three independent experiments were performed.

Exposures were performed in the Lab Tek Chamber slide system (Nalge Nunc International, Rochester, NY, USA) using 10⁶ cells/100 μL per well incubated with 6 mg/L of the different CNTs for 24 h. Next, the supernatant was removed and the cells were fixed and stained using fast Hemostain (Hycel, DF, Mexico). Photomicrographs were obtained using an Olympus BX4-1 microscope (Olympus, Miami, Florida, USA) equipped with a Pixera cold-coupled device camera (Pixera, Miami, Florida, USA) and analyzed with IMAGE Pro-Plus 4.1 software (Media Cybernetics, Silver Spring, Maryland, USA).

Human lung cell lines H441, H358, A549, H157 and H1299, were maintained in RPMI medium as previously described [20 (link)]. To examine the effect of hypoxia on phenotypic alterations in lung alveolar cell lines in vitro, the cells were cultured under hypoxia (1 % concentration of oxygen; 1 % O₂) for the indicated periods using a hypoxic chamber (Wakenyaku Co. Ltd., Tokyo, Japan) for the indicated periods. For immunocytochemistry, some cells were cultured in an 8-well Lab-Tek Chamber Slide System (Nalge Nunc International, Naperville, IL). Pimonidazole hydrochloride at final concentration of 150 μM was added 90 min before immunostaining for pimonidazole [6 (link)].