Unless otherwise stated, all cell imaging was done on M2G 1.5% agarose pads on a light-emitting diode-based (Lumencor, SpectraX) multicolor epifluorescence microscope consisting of a Leica Dmi8 stand equipped with an immersion oil phase contrast objective (100×, HC PL APO, 1.4 numerical aperture) and an EMCCD camera (Hamamatsu, C9100 02 Cl). Cells were grown overnight in M2G to an OD600 of 0.3–0.4. Images were analyzed and processed using Fiji software (68 (link)). For generating line profiles, the Fiji plugin, MicrobeJ (69 (link)), was used to divide cells into either 26 or 50 bins along the longitudinal axis, and the average fluorescence intensity of each bin was used to generate fluorescence profile plots. The protocol for imaging ϕCbK attachment was adapted from Hinz et al. (15 (link)). Briefly, Sytox Green (ThermoFisher S7020) was added to 1 mL of ϕCbK to a final concentration of 25 µM and allowed to incubate overnight at 4°C. Cells were mixed 1:1 with fluorescently labeled phage and imaged.
Dmi8 stand
Manufactured by Leica
The Dmi8 stand is a modular and versatile inverted microscope system developed by Leica. It features a robust and stable design to support a range of microscopy techniques and accessories. The Dmi8 stand provides a reliable and flexible platform for various imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Sytox green, by Thermo Fisher Scientific (3 mentions)
C9100 02 cl, by Hamamatsu Photonics (2 mentions)
Spectra x, by Lumencor (2 mentions)
Tcs spe confocal point scanner, by Leica (1 mentions)
Sp8 laser scanning confocal microscope, by Leica (1 mentions)
Orca flash4.0 v2, by Hamamatsu Photonics (1 mentions)
5 protocols using dmi8 stand
1
Multicolor Epifluorescence Microscopy of Phage-Host Interactions
2
Confocal Imaging of Immunofluorescence and miR FISH
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The Leica TCS SPE confocal point scanner mounted on a Leica DMi8 stand equipped with an oil immersion 63 × Apochromat was used in order to take confocal images of immunofluorescence and miR FISH samples. For all samples, the 405 and 561 nm laser lines were used to perform excitation. All images were analyzed using the ImageJ software and show one focal plane of the middle of the nucleus.
3
Multicolor Epifluorescence Microscopy of Immobilized Cells
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Cells immobilized on agarose pads or in vitro samples in multiwell glass bottom plates were imaged on a light-emitting diode–based (Lumencor, SpectraX) multicolor epifluorescence microscope consisting of a Leica Dmi8 stand equipped with an immersion oil phase contrast objective [100×, HC PL APO, 1.4 numerical aperture (NA)] and an EMCCD camera (Hamamatsu, C9100 02 CL). A list of all the filters used is provided in table S2.
4
Evaluating POTEE Overexpression in Breast Cancer
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The Kaplan–Meier (KM) plotter free database was used to evaluate how POTEE overexpression is associated with the evolution of BC patients [34 (link)]. Breast tumor studies were performed with the approval of The Bioethics Committee at the MD Anderson Cancer Center from Madrid (License number MD21/004) in accordance with the tenets of the Helsinki Declaration. Written informed consent was obtained from each patient before POTEE and Rac1 expression was evaluated in samples from different molecular subtypes of human tumors by immunofluorescence (IF). All samples were collected before 2021. IF staining was performed following the protocol described previously [35 ], and POTEE, Rac1 or POTEE‐Rac1 co‐localization was analyzed. Quantification of co‐localization between POTEE and Rac1 in the tumor samples was performed by analyzing five random areas. The number of cells showing co‐localization was divided by the total number of cells (identified by DAPI staining). This method allowed us to calculate the percentage of co‐localization for each tumor sample. Similarly, we quantified the individual expression of Rac1 or POTEE. We analyzed four different breast cancer tissues for each molecular subtype, with a total of four samples per subtype (N = 4). Immunofluorescence was analyzed under a Leica microscope (Leica SP8 Confocal Laser Scanning Microscope, with Dmi8 stand).
5
Single-molecule Localization Microscopy Setup
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Time lapse videos were acquired with a custom-built single-molecule localization microscope (SMLM) based on a Leica DMi8 stand with widefield illumination, a 63x/1.47 NA oilimmersion objective, and a band pass filter to collect emission fluorescence in the 584-676 nm wavelength range. Samples were illuminated at both 405 nm and 561 nm and imaged with an sCMOS camera (Hamamatsu ORCA-Flash4.0 V2) at 50 frames per second (fps) for 15,000 frames. To test different imaging conditions, the 405-nm laser intensity was set constant in a range from 0.5 to 2 W/cm 2 , and the 561-nm laser intensity from 1 W/cm 2 to 1 kW/cm 2 . A custom-written LABVIEW program was used to control the lasers for alternating 405-nm and 561-nm illumination.
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