Bacteria were grown overnight on BHI agar, resuspended in PBS and 20 µl spotted onto fresh BHI agar containing 1 mM IPTG and incubated for 4 hr at 37°C. After incubation, 500 µl of 109 CFU/mL bacterial suspension of attacker was mixed with the prey strain at a 1:1 ratio. Cells were harvested by centrifugation for 3 min at 6000 rpm, resuspended in 100 μL of PBS or GW media and 2 µl spotted on 1% agarose pads (for T6SS dynamics) or GW media with 0.1 mM IPTG and 0.5 µM SYTOXBlue (Thermo Fisher Scientific) for assessment of prey permeability. Fluorescence microscopy image sequences were acquired within 20–30 min of sample preparation with an inverted Zeiss 880 Airyscan microscope equipped with Plan-Apochromat 63×/1.4-NA oil lens and fitted with a climate chamber mounted around the objective to perform the imaging at 37°C with 5% CO2. Automated images were collected at 1 s, 10 s or 1 min intervals and processed with Fiji (Schindelin et al., 2012 (link)). Background noise was reduced using the ‘Despeckle’ filter. The XY drift was corrected using StackReg with ‘Rigid Body’ transformation (Thévenaz et al., 1998 (link)). Experiments and imaging were performed on at least two independent occasions.
Plan apochromat 63 1.4 na oil lens
Manufactured by Zeiss
The Plan-Apochromat 63×/1.4-NA oil lens is a high-performance objective lens designed for use in advanced microscopy applications. It features a high numerical aperture of 1.4 and a magnification of 63×, providing excellent image quality and resolution. The lens is optimized for use with oil immersion, allowing for improved light collection and increased contrast in samples.
Automatically generated - may contain errors
4 protocols using plan apochromat 63 1.4 na oil lens
1
Real-time Imaging of Type VI Secretion
2
Imaging of Fixed Immunofluorescence Samples
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Fixed cells on slides after immunofluorescence were imaged at 21°C using an inverted Zeiss 880 microscope fitted with an Airyscan detector using ZEN black software. The system was equipped with Plan-Apochromat ×63/1.4-NA oil lens, with an immersion oil (Immersol W 2010, Carl Zeiss; refractive index of 1.518). 488 nm argon and 405, 561, and 633 nm solid-state diode lasers were used to excite fluorophores. Z-sections with 0.37-μm-thick intervals were collected. The oil objective was covered with an immersion oil (ImmersolT 518F, Carl Zeiss) with a refractive index of 1.518.
Microscopy images with CZI file format were analyzed using ImageJ (bundled with Java 1.8.0_172) software. Scoring of nuclear morphology was done after maximum intensity projection image processing.
Microscopy images with CZI file format were analyzed using ImageJ (bundled with Java 1.8.0_172) software. Scoring of nuclear morphology was done after maximum intensity projection image processing.
3
Visualizing PLCγ1 and PDK1 Localization
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MDA-MB-231 cells were co-transfected with PRK5-PLCγ1 and pOZ-PDK1. Twentyfour hours after transfection cells were serum deprived overnight. The following day, cells were left untreated or treated with 50 μM 2-O-Bn-InsP5 for 30 minutes, stimulated with serum free DMEM containing EGF (50 ng/ml) in the presence or absence of 50 μM 2-O-Bn-InsP5 for the indicated times and then fixed in paraformaldehyde 4% (v/v) for 30 minutes at room temperature (RT). Cells were permeabilised with PBS containing 0.25% Triton-X-100 for 2.5 minutes at RT. Unspecific staining was prevented by blocking the coverslips with a solution of PBS/0.1% BSA for 30 minutes at RT. Coverslips were then incubated overnight with primary antibodies (anti-mouse-PLCγ1 and anti-rabbit-PDK1 diluted 1:50 in PBS/0.1% BSA). Coverslips were then washed 3X with PBS/0.1% BSA, incubated with secondary antibodies anti-mouse-Alexa488 (Life Technologies) and anti-rabbit Alexa555 (Life Technologies) and analysed using a Carl Zeiss LSM 510 Meta confocal microscope using a Zeiss plan apochromat 63× 1.4 NA(oil) lens.
4
Visualizing Bacterial Competition Dynamics
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Bacteria were grown overnight on BHI agar, resuspended in PBS and 20 µl spotted onto fresh BHI agar containing 1 mM IPTG and incubated for 4 h at 37°C. After incubation, 500 µl of 10 9 cfu/mL bacterial suspension of attacker was mixed with the prey strain at a 1:1 ratio. Cells were harvested by centrifugation for 3 min at 6000 rpm, resuspended in 100 μL of PBS or GW media and 2 µl spotted on 1% agarose pads (for T6SS dynamics) or GW media with 0.1 mM IPTG and 0.5 µM SYTOX™Blue (Thermo Fisher Scientific) for assessment of prey permeability. Fluorescence microscopy image sequences were acquired within 20-30 minutes of sample preparation with an inverted Zeiss 880 Airyscan microscope equipped with Plan-Apochromat 63×/1.4-NA oil lens and fitted with a climate chamber mounted around the objective to perform the imaging at 37°C with 5% CO2. Automated images were collected at 1 sec, 10 sec or 1 min intervals and processed with Fiji (Schindelin et al., 2012) .
Background noise was reduced using the "Despeckle" filter. The XY drift was corrected using StackReg with "Rigid Body" transformation (Thévenaz et al., 1998) . Experiments and imaging were performed on at least two independent occasions.
Background noise was reduced using the "Despeckle" filter. The XY drift was corrected using StackReg with "Rigid Body" transformation (Thévenaz et al., 1998) . Experiments and imaging were performed on at least two independent occasions.
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