Tcp sp8 confocal microscope

Laser micro-irradiation was performed on 1 million A9 cells cultured on glass bottom dishes (MatTek Corp.) infected with MVMp at an MOI of 10 for 18 hr. Cells were sensitized with 2 microliters of Hoechst dye (ThermoFisher Scientific) 5 min prior to irradiation. Samples were irradiated using a Leica TCP SP8 confocal microscope with a 405 nm laser using 25% power at 40 Hz frequency for 2 consecutive frames per field-of-view. Regions of interest (ROIs) were selected within the nucleus without traversing the nuclear membrane. Samples were processed for immunofluorescense imaging without CSK pre-extraction immediately after micro-irradiation.

12-well trays containing glass microscope coverslips were seeded with McCoy cells the day before the start of the experiment. Cells were infected with the appropriate amount of C. trachomatis A2497WT, A2497P-, or A2497P-/pSW2::GFP inoculum to give an MOI of 1. The 12 well trays were centrifuged and incubated at 37°C/5% CO₂ as described above. At each time point, cells were fixed using 4% paraformaldehyde for 15 min. Cells were washed in PBS and permeabilised in saponin buffer (0.1% saponin, 10% foetal calf serum, 0.1% sodium azide) for 1 h at 4°C. Monoclonal antibody MAb29 was added (in saponin buffer) at 1:1,000 dilution and cells were incubated for 1 h at room temperature. These were then washed three times in saponin buffer before the anti-mouse-Alexa-fluor 488 conjugate antibody (Fisher Scientific) was added at 1:200 dilution. This was again incubated for 1 h at room temperature, washed as before and counterstained with 1 μg/ml DAPI (Fisher Scientific) and Wheat Germ Agglutinin Alexa Fluor® 594 conjugate (Invitrogen), washed a final time in PBS and mounted onto slides with Mowiol mounting medium (Sigma Aldrich). Images were captured using a Leica TCP SP8 confocal microscope. Areas of inclusions were measured at each time point using ImageJ (version 1.51j8) and compared using a one-way ANOVA using GraphPad Prism (version 7.03).

MDA-MB-231 (5.0 × 10⁴ cells/well) and MCF-7 (5.0 × 10⁴ cells/well) cells were seeded on confocal dishes (SPL Life Sciences Co., Seoul, Korea) and cultured with different concentrations of GLE (0, 50, 100, and 200 μg/mL) in the incubator. After 24 hours, medium was replaced with a live staining cocktail of 10 μM 2′7′-dichlorofluorescein diacetate (denoted as DCF-DA, ≥ 95%, Sigma), 50 μg/ml propidium iodide (≥ 94.0%, Sigma), and 1 μg/mL bisBenzimide H 33342 trihydrochloride (≥ 97.0%, Sigma). After 20 minute incubation with fluorophores, cells were washed twice and stained cells were observed on a TCP SP8 confocal microscope (Leica, Germany).

Cells were grown on cover slips in dishes. Following treatment, the cells were fixed with 4% paraformaldehyde (in PBS) for 15 min. The fixed cells were blocked with 1% BSA (in PBS) containing 0.1% Triton X-100 for 30 min at room temperature. The cells were incubated with the indicated primary antibodies at 4 °C overnight. Alexa Fluor^® 568 Phalloidin was obtained from Invitrogen and anti-Tks5 was purchased from Santa Cruz Biotechnology. Following an overnight incubation, the cells were incubated with the corresponding fluorescent-conjugated secondary antibodies for 2 h at room temperature. DAPI (0.5 μg/ml; Vector Laboratories, Burlingame, CA, USA) was used to counterstain the nuclei. The images were obtained using a Leica TCP-SP8 confocal microscope (Leica, Wetzlar, Germany) and Zeiss ApoTome microscope (Carl Zeiss, Jena, Germany). The co-localization rate and Pearson’s co-localization coefficients were determined with LAS AF Image software (Leica). Co-localization rate is the value that indicates the extent of co-localization in percentage and is calculated as follows: 100 × (Co-localization Area/Area Foreground), where area foreground is the difference between total area of the image and area of the image background. The values of Pearson’s coefficients ranging from 0.5 to 1 are considered positive co-localization.