Lab tek 2 chamber slide system

MCF7 cells were plated in 8-well chambers (Nunc^™ Lab-Tek^™ II Chamber Slide^™ System, Cat #154453), treated with vehicle or 40 μM imipramine for 60 h, and then fixed with 4% PFA for 20 min at room temperature. The fixed cells were blocked and permeabilized with 5% normal goat serum in 0.1% Triton X-100 for 30 min at room temperature, and then incubated with 53BP1 rabbit antibody (1:500 dilution, Bethyl Laboratories, Cat #A300-272AT) overnight at 4 °C. Images were captured using an Olympus microscope under 100x magnification.

Intracellular generation of ROS in hRPE and ARPE19 cells as the result of HOHA lactone treatment was evaluated using DCFHDA according to the method of Wang and Joseph[29 (link)] with minor modifications. Briefly, either hRPE or ARPE19 cells (45,000 cells/ per well) were plated on 8-chamber well (Lab-Tek II Chamber Slide System, Nunc, Rochester, NY) in the respective complete medium (with 10% FBS). The following day, the cells were starved in a basal medium for 4–5 hours. Cells were pre-treated with DCFH-DA for 45 minutes with 13.3 μM DCFH-DA in the respective basal medium at 5% CO₂/95% air at 37 °C. The cells were washed, and then further treated with 0, 15, or 30 μM of HOHA lactone solution for 30 minutes at 5% CO₂/95% air at 37 °C. The images were taken at 10X magnification with a Leica DMI 6000 B fluorescence inverted microscope.

Either ARPE-19 cells or hRPE cells (2.5 × 10⁴ cells/ per well) were plated on an 8-chamber slide (Lab-Tek II Chamber Slide System, Nunc, Rochester, NY) in the corresponding complete medium (with 10% FBS) at 37 °C and 5% CO₂ overnight. After starving the cells in the corresponding basal medium overnight, the cells were washed three times with the corresponding basal cell culture medium. Then the cells were exposed to 0 or 25 μM H₂O₂ in PBS buffer for 2 h at 37 °C and 5% CO₂ The cells were aspirated and washed three times with the corresponding basal cell culture medium followed by overnight incubation at 37 °C and 5% CO₂. Alternatively, the cells were exposed to 0 or 12 μg/mL LPS in PBS buffer overnight at 37 °C and 5% CO₂. After incubation, the slides were centrifuged at 500g for 5 min and the medium was aspirated from each chamber. Cells were fixed with dry-ice cold acetone and immunostaining was performed as described above for HOHA-lactone treated RPE cells. Images were taken at 10X magnification with a Leica DMI 6000 B fluorescence inverted microscope. Image analysis was performed using Metamorph imaging software (Molecular Devices, Downington, PA).

Detection of enhanced annexin V binding to ARPE19 cells as the result of HOHA lactone treatment was achieved using the CF488A-annexin V - propidium iodide apoptosis assay kit (Biotium, Hayward, CA). ARPE 19 cells (20,000 cells/ per well) were plated in 8-chamber wells (Lab-Tek II Chamber Slide System, Nunc, Rochester, NY) in complete DMEM/ F12 medium (10%FBS) and allowed to attach to the slide’s surface overnight in 5% CO₂/95% air at 37 °C. The following day, the cells were starved in basal DMEM /F12 medium for 4–5 h. The cells were then incubated overnight (16 h) with 0 and 15 μM HOHA lactone in 5% CO₂/95% air at 37 °C. Following incubation with HOHA lactone, the cells were stained with a mixture of annexin V/propidium iodide following the protocol described in manufacturer’s manual. Images were taken at 10X magnification with a Leica DMI 6000 B fluorescence inverted microscope. Image analysis was performed using Metamorph imaging software (Molecular Devices, Downington, PA).