The growth analysis was performed in S. cerevisiae acl4Δ strains that were transformed with pRS415 constructs carrying various mCherry-tagged Acl4 variants. Transformed cells were selected twice on SDC-LEU plates, before analysis. Ten-fold dilution series were prepared and 17.5 μl were spotted on SDC-LEU plates and grown at 23, 30 and 37 °C for 2–3 days. Localization assays were performed using pRS415 vectors carrying mCherry-tagged Acl4 variants and a pRS413 vector harbouring eGFP-tagged RpL4. Transformed cells were selected twice on SDC-LEU-HIS plates before analysis. The variants were grown in SDC-LEU-HIS medium at 30 °C to mid-log phase. For heat-shock analysis, cells were grown at 30 °C to mid-log phase before shifting cells to 37 °C for 6 h. For fluorescence microscopy 1 ml of cells was centrifuged at 500g and washed once with 1 ml of water. The cell pellet was resuspended in 100 μl water and 10 μl were analysed using a Carl Zeiss Observer Z.1 equipped with a Hamamatsu C10600 Orca-R2 camera.
C10600 orca r2 camera
Manufactured by Hamamatsu Photonics
Sourced in Japan
The C10600 Orca-R2 camera is a high-performance scientific camera designed for a variety of imaging applications. It features a high-resolution, low-noise sensor and advanced image processing capabilities. The camera is capable of capturing images with high spatial and temporal resolution.
Automatically generated - may contain errors
Lab products found in correlation
Observer z 1, by Hamamatsu Photonics (1 mentions)
Axiovision 4, by Zeiss (1 mentions)
Fm4 64, by Thermo Fisher Scientific (1 mentions)
Fm4 64, by Zeiss (1 mentions)
Microbej, by Zeiss (1 mentions)
Axio observer microscope, by Zeiss (1 mentions)
Plan apochromat, by Zeiss (1 mentions)
Las af 6000, by Leica (1 mentions)
Ti inverted microscope, by Nikon (1 mentions)
Ultraview vox spinning disk confocal system, by PerkinElmer (1 mentions)
Hibernate a low fluorescence medium, by Transnetyx (1 mentions)
Ctr7000 hs, by Leica (1 mentions)
Wskm stage top incubator, by Tokai Hit (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Concanavalin a (cona), by Merck Group (1 mentions)
Axioplan fluorescence microscope, by Zeiss (1 mentions)
Axio imager m2 microscope, by Zeiss (1 mentions)
7 protocols using c10600 orca r2 camera
1
Acl4 Variant Localization Analysis
2
Visualization of Bacterial Cell Division
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Cells were collected in exponential phase from MRS cultures (with antibiotics and inducers when needed) and resuspended in PBS buffer. Bacteria were observed on agarose pads composed of 1% agarose PBS buffer for static observations and of 1% agarose MRS for time-lapse observations. Cellular membranes were stained with FM4-64 (Life Technologies) as reported before (Andre et al., 2011 (link)). Images were obtained using an Axio I inverted microscope (Zeiss) equipped with an α Plan-Apochromat objective (100 × /1.46 Oil DIC M27) (Zeiss), a HXP 120 C lighting unit (Zeiss) and C10600 ORCA-R2 camera (Hamamatsu). The fluorescence of FM4-64 and FtsZ-GFP+ was respectively detected with filter sets Cy3 (43 HE) and GFP (38 HE), displaying bandpass excitation (nm): 550/25 (Cy3) or 470/40 (GFP) and bandpass emission: 605/70 (Cy3) or 525/50 (GFP) (Zeiss). Images were analyzed using Axiovision 4.8 (Zeiss), MicrobeTracker (Sliusarenko et al., 2011 (link)), or MicrobeJ (Ducret et al., 2016 (link)).
3
Visualizing Protein-Protein Interactions in βTC3 Cells
4
Live-Cell Imaging of Axon Dynamics
5
Time-lapse Fluorescent Microscopy of Phagocytosis
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For the video of the time-lapse fluorescent microscopy of phagocytosis, 2 × 104 or 1 × 105 THP-1 cells were plated into flat-bottom, polylysine-coated, 96-well plates (LabTek, MatTek). Then, 100,000 or 500,000 yellow or nile blue (CFL-5065-2; 5.0-5.9μm, Spherotech) fluorescent beads coupled with VAR2CSA or KLH were added to the adherent THP-1 cells. Phagocytosis was immediately imaged using an automated IX-81 microscope (Olympus, Japan) with a WSKM stage top incubator (Tokai Hit, Japan) that kept the cells at 37 °C and 5% CO2. Cells were imaged at a magnification of 320X every 30 seconds for 1 hour. For each time point, three images were taken in sequence, one for bright field, one using a GFP filter set, and one using a TxRed filter set. All images were captured using an Orca-R2 C10600 camera (Hamamatsu, Japan) controlled by Metamorph v.7.8.1 software (Molecular Devices, Downington, PA) which combined the images collected every 30 seconds into the video.
6
Imaging C3 Cells with Chemical Inducers
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C3 cells were grown on 13 mm No. 1 round coverslips (VWR) coated with Concanavalin-A (Sigma-Aldrich) and allowed to adhere over night. Cells were incubated in the presence of AP21998 or D/D solubilizer for 80 minutes. Cells were then fixed and stained as described in [62 (link)]. Coverslips were mounted using ProLong Gold (Molecular Probes Invitrogen) and sealed with clear nail polish. Images were captured using either a 63x or 100x oil objective on a Zeiss Axioplan fluorescence microscope (Zeiss) equipped with a Hamamatsu Orca-R2 C10600 camera (Hamamatsu Photonics), and SEDAT quad pass filter set (Chroma). The brightness and contrast of microscopy images were adjusted using ImageJ (NIH).
7
Visualizing Live and Dead Borrelia burgdorferi
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Specimens were examined on a Zeiss AxioImager M2 microscope equipped with differential interference contrast (DIC) and epifluorescent illumination and were recorded with a Hamamatsu ORCA-R2 C10600 camera. A cell proliferation assay was performed by direct counting using a bacterial counting chamber (Hausser Scientific Partnership, Horsham, PA, USA) and DIC microscopy. A SYBR Green I/PI assay was performed to assess the viability of B. burgdorferi, as previously described.13 (link) The ratio of live (green) and dead (red) B. burgdorferi was calculated by counting the cells using a bacterial counting chamber and epifluorescence microscopy, as previously described.13 (link) For the aggregated cells, three representative images of each sample were captured for quantitative analysis. Image Pro-Plus software was applied to select the green (including yellow) and red (including orange) areas of different morphological forms to calculate the integrated fluorescence intensity (equal to area × average density or average intensity) of the red and green portions, as previously described.16
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