BMDM were plated on a 96 well glass bottom plate with #1.5 cover glass and a black frame (CellVis) at 1×105 cells per well in I-10 media overnight. The cells were stained with 5 μM CellTracker™ Orange CMTMR Dye for 37 °C x 30 min then washed. Cells were washed and incubated in 150 μL I-10 media. 50 μL of a 1:31 dilution of single CPS-expressing B. theta strains that endogenously expressed GFP with or without 10 μg/mL of B. theta antibody was added to each well. B. theta strains expressing a single CPS and GFP were grown in a 2mL TYG culture at 37 °C overnight to mid log phase. Cultures were washed once and resuspended in PBS prior to adding to the assay. Four hours later, images were acquired in the same z-plane using an Olympus IX70 microscope with a 100x/1.4NA oil objective, a Yokogawa spinning-disk confocal scanning unit, and a Hamamatsu Orca Flash4 CMOS camera. Cells were maintained at 37 °C with 5.0% CO2 in a Tokai Hit humidified chamber. Images were acquired with NIS-Elements AR software and the number of bacteria uptaken per BMDM was measured manually using Imaris software. A total of 150 individual cells were measured for each bacterial strain analyzed with 50 cells measured per experiment in 3 experiments.
Ix70 microscope
Manufactured by Yokogawa
The IX70 is a high-performance inverted microscope designed for advanced microscopy applications. It features a stable and vibration-resistant frame, a wide range of observation methods, and user-friendly operation. The IX70 provides researchers with a versatile platform for various microscopy techniques.
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Lab products found in correlation
Orca flash4 cmos camera, by Hamamatsu Photonics (2 mentions)
Spinning disk, by Hamamatsu Photonics (1 mentions)
Eclipse ti microscope, by Yokogawa (1 mentions)
Csu x1 confocal scanner, by Yokogawa (1 mentions)
Ultraview vox confocal imaging system, by PerkinElmer (1 mentions)
Dmi8 microscope, by Leica camera (1 mentions)
Csu x1 spinning disk confocal, by Yokogawa (1 mentions)
Metamorph, by Molecular Devices (1 mentions)
Rat tail collagen 1, by BD (1 mentions)
4 protocols using ix70 microscope
1
Visualizing B. theta Uptake by BMDMs
2
Fluorescence Imaging of Protein Aggregation
3
Confocal Microscopy Imaging of Dissociated Cultures and Brain Sections
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Unless otherwise indicated, all fluorescence images shown in this article represent maximum projection images derived from a z-stack.
To acquire images of fixed dissociated cultures or brain sections we used an Olympus IX-70 microscope equipped with a CSU-X1 spinning disk confocal (Yokogawa Electric Corporation) custom equipped with 405 nm, 491 nm, 561 nm and 640 nm 50 mW solid state lasers (Solamere Technology Group Inc.) and a CoolSNAP HQ2 digital CCD camera (Photometrics) with pixel size of 91 nm. Fluorescence emission was selected through the following bandpass filters: 525/50 nm, 595/50, 700/75. Metamorph (Molecular Devices) was used to acquire a stack of 6–11 images in the z-dimension using optical slice thickness of 0.2 µm for 60X images of dissociated cultures and brain sections. A single plane of focus was used to acquire low magnification images of brain sections using a ×1.25 objective.
To acquire images of fixed dissociated cultures or brain sections we used an Olympus IX-70 microscope equipped with a CSU-X1 spinning disk confocal (Yokogawa Electric Corporation) custom equipped with 405 nm, 491 nm, 561 nm and 640 nm 50 mW solid state lasers (Solamere Technology Group Inc.) and a CoolSNAP HQ2 digital CCD camera (Photometrics) with pixel size of 91 nm. Fluorescence emission was selected through the following bandpass filters: 525/50 nm, 595/50, 700/75. Metamorph (Molecular Devices) was used to acquire a stack of 6–11 images in the z-dimension using optical slice thickness of 0.2 µm for 60X images of dissociated cultures and brain sections. A single plane of focus was used to acquire low magnification images of brain sections using a ×1.25 objective.
4
Imaging and Analyzing Macrophage Podosome Dynamics
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