Phase-contrast microscopic images of unstrained and strained hMSCs were obtained (Olympus, Japan) in at least four randomly selected sites from our visual field. To observe the effect of cyclic loading on cytoskeletal actin arrangements, hMSCs at all conditions were stained with fluorescent phallotoxins (Molecular Probes, Oregon, USA) for 30 min and then the nucleus stained with Hoechst (Molecular Probes, Oregon, USA) for 10 min in the dark. Fluorescence was recorded using a laser scanning confocal attachment (Leica TCS SP5 II, Germany) and measured by LAS AF image software (Leica, Germany). Images of unstrained MSCs on silicone membrane served as control.
Las af image software
Manufactured by Leica
Sourced in Germany
LAS AF is a comprehensive image processing software designed for use with Leica microscopy systems. The software provides essential tools for image acquisition, analysis, and visualization, supporting a wide range of microscopy techniques and file formats.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescent phallotoxins, by Thermo Fisher Scientific (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Tcs sp5 2, by Leica (1 mentions)
Dm5500q confocal microscope, by Leica (1 mentions)
Sp8 x inverted confocal microscope, by Leica (1 mentions)
Prism v7, by GraphPad (1 mentions)
Prism 7, by GraphPad (1 mentions)
Apotome microscope, by Zeiss (1 mentions)
Tcp sp8 confocal microscope, by Leica (1 mentions)
Anti tks5, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 568 phalloidin, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Dm1000 microscope, by Leica (1 mentions)
Dcf350fx, by Leica (1 mentions)
Dm5500b, by Leica (1 mentions)
6 protocols using las af image software
1
Cytoskeletal Actin Dynamics in Strained hMSCs
2
Confocal Microscopy Z-Stack Imaging
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Images were acquired using a Leica DM5500Q confocal microscope. Leica LAS AF image software was used to take a series of XY optical sections, 1.0 μm apart, throughout the depth of the section and built into a stack to give a projection image.
3
Imaging Aspergillus fumigatus Infection Dynamics
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Cells were seeded at 2 × 105 cells per well in a 24-well glass bottom plates (Corning, UK) and incubated for 16 h until confluent. Cells were serum-starved for 48 h and then infected with 2 × 104 spores from the A. fumigatus strains described in Supplementary Table 3 . The plate was then mounted under a Leica SP8X inverted confocal microscope using a 40 × dry lens objective in a 5% CO2 environment at 37 °C. Three different positions per well in 3 independent biological and technical replicates were evaluated. Images were captured using the brightfield transmitted light detector of the microscope every 30 min for 11 h driven by the Leica LASAF image software. The videos generated by this software were exported as AVI documents and processed with Image J software (NIH) (http://rsb.info.nih.gov/ij ). Hyphal length, A. fumigatus germination time point and total growth were measured using Fiji Software (https://fiji.sc/ ). Differences in A. fumigatus hyphal length and germination time point were determined by T-test at fixed time points after confirming normal distribution (GraphPad Prism v7). Differences in A. fumigatus growth curves were calculated using the non-parametric Kruskal–Wallis test using GraphPad Prism 7 software. Experiments were performed in biological and technical triplicates.
4
Immunofluorescence Assay for Tks5 Colocalization
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Cells were grown on cover slips in dishes. Following treatment, the cells were fixed with 4% paraformaldehyde (in PBS) for 15 min. The fixed cells were blocked with 1% BSA (in PBS) containing 0.1% Triton X-100 for 30 min at room temperature. The cells were incubated with the indicated primary antibodies at 4 °C overnight. Alexa Fluor® 568 Phalloidin was obtained from Invitrogen and anti-Tks5 was purchased from Santa Cruz Biotechnology. Following an overnight incubation, the cells were incubated with the corresponding fluorescent-conjugated secondary antibodies for 2 h at room temperature. DAPI (0.5 μg/ml; Vector Laboratories, Burlingame, CA, USA) was used to counterstain the nuclei. The images were obtained using a Leica TCP-SP8 confocal microscope (Leica, Wetzlar, Germany) and Zeiss ApoTome microscope (Carl Zeiss, Jena, Germany). The co-localization rate and Pearson’s co-localization coefficients were determined with LAS AF Image software (Leica). Co-localization rate is the value that indicates the extent of co-localization in percentage and is calculated as follows: 100 × (Co-localization Area/Area Foreground), where area foreground is the difference between total area of the image and area of the image background. The values of Pearson’s coefficients ranging from 0.5 to 1 are considered positive co-localization.
5
GnRHR-Arrestin Interaction Imaging
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HEK 293 cells were plated on poly-d -lysine (1 mg/ml)-coated coverslips, placed in a 24-well plate. The next day, cells were transfected with 300 ng GnRHR-WT or GnRHR-Ctail along with 200 ng of pYFP-Arrestin. Twenty-four- to 48-hr post-transfection, cells were treated with 100 nM GnRH or 1 μM of the GnRH analog Lucrin for 1, 5, or 20 min or left untreated as control. Cells were then fixed for 20 min with fresh 4% PFA and 0.2% Triton X-100. Coverslips were than mounted on glass slides using ProLong Gold. Fluorescence images were acquired with a Leica DM-1000 microscope with a ×40 objective, or confocal microscope (Leica SP5) with a 63 × 1.4 numerical aperture objective. Leica LAS AF image software was utilized for image acquisition.
6
Quantifying Mitotic and Adhesion Phenotypes
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Mitotic and adhesion phenotypes were quantified by epifluorescence microscopy under a fluorescent (DM5500B; Leica) microscope using a 20×/NA 0.15 objective lens at room temperature. All antibodies used for immunofluorescence were employed according to manufacturer’s instructions (a complete list is reported in the Supplemental Experimental Procedures ). Fluorochromes used were Cy3 (cyclin B, lamin B, vinculin, pMLC2, Flag-tag), Alexa488 (pH3), FITC-/TRITC-phalloidin, or Cy5 (vinculin [pH3], Myc-tag). All images were acquired with a camera (DCF350FX; Leica) and LAS-AF image software (Leica). Images were processed with the same settings (brightness, contrast, crop, image size) using Adobe Photoshop CS5.1, and imaged figures were constructed in Adobe Illustrator.
Confocal analysis of DEPDC1B localization, actin cytoskeleton, and FAs was performed on a Leica TCS SP2 AOBS microscope, using a 40×/NA 1.25 oil-immersion objective and processed in Adobe Photoshop. Images were taken with identical settings, and the number and average area of FAs per cell were determined using ImageJ software with a mask with a fixed threshold that identifies vinculin- or paxillin-GFP positive FAs. Details on live microscopy (time-lapse and TIRF) are in theSupplemental Experimental Procedures .
Confocal analysis of DEPDC1B localization, actin cytoskeleton, and FAs was performed on a Leica TCS SP2 AOBS microscope, using a 40×/NA 1.25 oil-immersion objective and processed in Adobe Photoshop. Images were taken with identical settings, and the number and average area of FAs per cell were determined using ImageJ software with a mask with a fixed threshold that identifies vinculin- or paxillin-GFP positive FAs. Details on live microscopy (time-lapse and TIRF) are in the
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