Chamber slide

The brains of 10-day-old cockatiel embryos were removed with a 10-mL syringe that contained 2 mL of Dulbecco’s Modified Eagle’s medium (DMEM, Gibco, Invitrogen, Life Technologies Ltd., Paisley, UK) with 10% fetal calf serum (FCS) through a hypodermic needle (20 G × 1.5 inches). The brain suspension was added to 1 mL of DMEM with 10% FCS onto chamber slides (Nunc A/S, Roskilde, Denmark). The medium was changed after 2 days of incubation at 37 °C. Inoculation with PaBV-4 or PaBV-2 was performed after the monolayer showed 75% confluence. The cells were inoculated with 10-fold serial dilutions in medium containing 10% FCS. Every dilution was incubated on chamber slides for 6 days at 37 °C.

Ros 17/2.8 cells and MG63 cells were plated on chamber slides (NalgeNunc International, Naperville, IL, USA). When the cells were extended thoroughly, they were washed three times with PBS and fixed with 4% paraformaldehyde in PBS for 25 min. Following three washes with PBS, the cells were permeabilized with 0.1% Triton X-100 for 5 min at room temperature. Then they were blocked with 1% BSA for 1 h at room temperature followed by three washes with PBS. The cells were incubated with anti-actinin 4 antibody (Abcam, Cambridge, MA, USA) and anti-β₃ subunit antibody (Alomone, Jerusalem, Israel) at 4°C over night and 30 min at room temperature the next day with gently rocking. After washing with PBS for 10 min for three times, the cells were incubated with the secondary antibodies with labeled with Rhodamine or FITC (Jackson Lab., Barharbor, Maine, USA) at room temperature for 2.5 h shield from light. After three washes with PBS likewise, cells were counterstained with DAPI nuclear stain for 5 min and washed once with PBS, and mounted with Vectashield and reserved at 4°C shield from light. The fluorescent signals were visualized by confocal microscopy (LSM 410; Carl Zeiss, Oberkochen, Germany).The co-localization of actinin 4 and L-type calcium channel β₃ subunit was determined by evaluating at least five samples.

Apoptotic cells were detected by TdT-mediated dUTP nick end labeling (TUNEL). The cells were plated on the chamber slides (Nunc, Roskilde, Denmark) at a concentration of 10⁵ cells/slide and cultured with or without genotoxic treatments such as serum-free medium or 250 μM hydrogen peroxide treatments. Cells were then fixed with 4% PFA for 15 min. After washing with PBS, the slides were treated with 0.3% hydrogen peroxide to block endogenous peroxidase. After washing with PBS, TUNEL staining was performed using the in-situ Apoptosis Detection kit (Takara) which label 3′-OH ends of fragmented DNA with fluorescein dUTP and visualized under an Eclipse TE300 fluorescence microscope. The degree of apoptosis was calculated by the percentage of TUNEL positive signals. For each cell line, at least 97 cells were evaluated, and three TUNEL experiments were analyzed. Only nuclear positive signals were used for analysis. Apoptotic cells were also detected by a flow cytometer (MACSquant) using Annexin V-fluorescein isothiocyanate (FITC) and propidium iodine (PI), which detect Annexin V-accessible internal phosphatidylserine and permeabilized cell membrane, respectively.

Immunofluorescence staining was performed on chamber slides (Nalge Nunc International, Naperville, IL). JEG-3 cells or human placental tissue slides were fixed in ice-cold 4% paraformaldehyde (PFA) and permeabilized with 0.1% Triton X-100 in PBS (PBST). After incubation with blocking buffer (1% bovine serum albumin, BSA), samples were incubated with primary antibodies against aromatase and RGS2 and were subsequently incubated with Alexa 546-conjugated and Alexa 488-conjugated secondary antibodies (Life Technologies), respectively. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). Slides were analyzed by a laser scanning microscope (Zeiss).

Apoptosis was confirmed by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis using an in situ cell death detection kit (Roche, Mannheim Germany). Cells were seeded (30,000 cells) on chamber slides (Nunc, Denmark) and treated with 300 μg/ml IV extract. After 48 and 72 h, cell morphology was examined using 4′,6-diamidino-2-phenylindole (DAPI) and TUNEL staining. At the end of treatment, cells were washed twice with PBS, fixed for 60 min with 4% paraformaldehyde and then permeabilized, using 0.1% Triton X-100 in 0.1% sodium citrate, to allow penetration of the TUNEL reaction reagents into the cell nucleus. TUNEL reaction mixture (TdT and fluorescein-dUTP) was added to label the fragmented DNA at 37°C for 1 h in humidified atmosphere in dark. After incubation time, cells were washed twice in PBS, and stained with DAPI solution in order to assess total cell number and for visualization of DNA morphology. Finally, the labeled DNA and the nucleus area were visualized by fluorescence microscopy (Nikon, Kawasaki, Japan).

Human lung epithelial cells (BEAS-2B, ATCC, Manassas, VA) were seeded at 2.5 × 10⁵ cells/slide in chamber slides (Nunc, Naperville, IL), incubated for 30 min at 37 °C with FITC-labeled HNP (10 μg/mL). In additional chamber slides, BEAS-2B cells were treated for 30 min with 10 μM of reactive blue (a purinoceptor (P2Y) antagonist) [23 (link)], or suramin (a blocker for receptor-mediated endocytosis), prior to HNP stimulation (100 μg/mL). The cells were then fixed, stained with rhodamine-phalloidin (ThermoFisher Scientific), a high-affinity F-actin probe conjugated to the red-orange fluorescent dye for F-actin, and mounted for visualization.
In separate experiments, BEAS-2B cells or human monocytes (THP-1, ATCC, Manassas, VA) were treated with 10 μM of reactive blue or suramin 30 min before HNP stimulation (100 μg/mL) for 8 h. The supernatants were collected for measurement of IL-8 (MyBioSource, San Diego, CA).

Peritoneal macrophages from untreated mice were cultured overnight on chamber slides (Nalge Nunc International) in DMEM containing 10% FBS and 100 U/ml penicillin/streptomycin. The cells were serum-starved for 12 hours and subsequently treated with vehicle control (PBS contained 0.1% DMSO), lanatoside C (10 μM) or oxLDL (50 μg/ml) for an additional 24 hours. The non-adherent cells were washed away and the adherent cells were collected for Oil-red-O staining or cholesterol measurement. Upon fixation with paraformaldehyde (4%), the cells were stained with 0.3% oil red O for 15 min, Hematoxylin was used as a counterstain, and evaluated via microscopy. The stained cells were eluted with isopropanol, and the supernatant was collected; the OD of the extracts was measured at 540 nm.