Thunder imager 3d cell culture microscope

Mammospheres were stained with 2 μM JC-1 (Thermo Fisher Scientific) for 30 min at 37°C. JC-1 stained mammospheres were mixed with Matrigel, placed on 35 mm glass-bottom dishes and analyzed using a Leica THUNDER Imager 3D Cell Culture microscope. For flow cytometry analysis, JC-1-stained mammosphere cells were washed and labeled with Zombie Aqua™ (BioLegend). Emission spectral overlap was corrected by compensation using carbonylcyanide m-chlorophenylhydrazone (CCCP). Cells were analyzed by MACSQuant Analyzer 10 Flow Cytometer (Miltenyi Biotec). A total of 20,000 events were acquired for each sample. Data were analyzed with FlowJo v10.6.2 (BD Biosciences) software. Fluorescence was measured using the standard emission filters for green (FL-1 channel) red (FL-2 channel) fluorescence photomultipliers.

BT-549 and MDA-MB-436 cells were fixed in 4% paraformaldehyde (Sigma) at room temperature for 10 min. The samples were incubated with 0.1% Triton X-100 (Sigma) at room temperature for 10 min, blocked with 3% Normal Goat Serum (Gibco), incubated with anti-γH2AX (#9718, 1:400 dilution; Cell Signaling Technology) at 4 °C overnight and then incubated with goat anti-rabbit IgG H and L labeled with Alexa Fluor 488 (Abcam) at room temperature for 1 h. Invitrogen™ ProLong™ Gold Antifade Mountant with DAPI (Invitrogen) was used for staining of nuclei. The cells were analyzed using a Leica THUNDER Imager 3D Cell Culture microscope.

The Magic Red Cathepsin B Kit (#ICT938; Bio-Rad) was used to quantify and monitor cathepsin B activity in RPTEC/TERT1 cell lines. Briefly, differentiated control, CLCN5 KD, and ClC-5 rWT RPTECs were treated with either DMSO (vehicle) or bafilomycin A1, as a positive control, for 24 h. Next day, cells were washed three times with isotonic solution containing 2.5 mM KCl, 140 mM NaCl, 1.2 mM CaCl₂, 0.5 mM MgCl₂, 5 mM glucose, and 10 mM Hepes (305 mosmol/litre, pH 7.4 adjusted with Tris), and incubated with Magic Red cathepsin B reactive at 1:26 (vol/vol) for 5 min at 37°C (following the manufacturer’s protocol). After the incubation period, the cells were imaged using Leica Thunder Imager 3D Cell Culture Microscope. All data were processed and analysed using FIJI (47 (link)).

Mammospheres were fixed with 4% paraformaldehyde (Sigma-Aldrich) at room temperature for 15 minutes. Samples were incubated with 1% Triton X-100 (Sigma-Aldrich) at room temperature for 10 minutes and blocked with 5% normal goat serum (Gibco). The mammospheres were attached to slides via cytospin at low speed (Shandon Cytospin 3; Shandon Scientific) and stained with anti-γH2AX (#9718, 1:400 dilution, CST) and goat anti-rabbit IgG H and L labeled with Alexa Fluor 488 (Abcam) as described (41 (link)). Nuclei were stained with ProLong Gold Antifade Mountant with DAPI (Invitrogen). Cells were imaged using a Leica THUNDER Imager 3D Cell Culture microscope, as described (41 (link)).

Spore titres of relevant strains were determined by standard techniques. S. coelicolor (10⁷ colony forming units (c.f.u.s)) of strains WT, SS387, SS393, SS395 and SS540 were spread onto R2YE agar plates and grown at 30 °C. Sterile glass coverslips were gently applied to the top surface of each bacterial lawn after 48 h, 72 h and 96 h post inoculation. Cover slips were then mounted onto glass microscope slides and imaged using a ×40 objective on a Leica Thunder Imager 3D cell culture microscope. Images were processed using FIJI⁵¹ .

Cells were incubated with pre-warmed MitoSOX Red staining solution diluted in HBSS to a final concentration of 5 μM for 15 min at 37°C. MitoSOX Red stained mammospheres were mixed with Matrigel, placed on 35 mm glass-bottom dishes and analyzed using a Leica THUNDER Imager 3D Cell Culture microscope. For flow cytometry analysis, MitoSOX Red-stained mammosphere cells were washed and labeled with Zombie Aqua™ (BioLegend). Cells were analyzed by MACSQuant Analyzer 10 Flow Cytometer (Miltenyi Biotec). A total of 20,000 events were acquired for each sample. Data were analyzed with FlowJo v10.6.2 (BD Biosciences) software. Fluorescence was measured using the standard emission filters for red (FL-2 channel) fluorescence photomultipliers.