The glioblastoma MCTS (LN-229) were grown as described above. For fluorescence microscopy, PI staining was used to determine the toxicity of the peptides implied by induction of membrane damage and cell death. Therefore, the MCTS were treated with peptides in different concentrations (2 µM, 5 µM, 10 µM and 20 µM) for 24 h. Then MCTS were harvested using Corning® DeckWorks low binding pipet tips from Sigma-Aldrich (Deisenhofen, Germany), released onto ibidi 60 µ-Dish with glass bottom (Martinsried, Munich, Germany) and stained with 2 µL of a 50 µg/µL PI solution (in DPBS buffer) per spheroid. The experiments were performed with a Leica DMI6000 B with IMC in combination with a Leica DFC360 FX camera and AF 6000 software (Leica Microsystems, Vienna, Austria). For microscopic inspection, the excitation wavelength for PI was set to 538 nm, and the emission wavelength was set to 617 nm.
Leica dfc360 fx camera
Manufactured by Leica Microsystems
Sourced in Germany
The Leica DFC360 FX is a high-performance digital camera designed for microscopy applications. It features a scientific-grade CMOS sensor that delivers high-resolution images with excellent color accuracy and low noise. The camera is compatible with a wide range of Leica microscopes and can be used for various imaging tasks, including documentation, analysis, and publication.
Automatically generated - may contain errors
Lab products found in correlation
Af6000, by Leica Microsystems (4 mentions)
Las af, by Leica Microsystems (3 mentions)
Leica dm6000b fluorescent microscope, by Leica Microsystems (3 mentions)
Leica dmi6000 b with imc, by Leica camera (2 mentions)
Normal goat serum (ngs), by Abcam (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Agilent Technologies (2 mentions)
Triton 100, by Merck Group (2 mentions)
Rabbit polyclonal igg, by BioLegend (2 mentions)
Bovine serum albumin (bsa), by BioLegend (2 mentions)
Vectashield containing dapi, by Vector Laboratories (2 mentions)
Leica dmi4000 inverted microscope, by Leica camera (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Leica dfc295, by Leica camera (1 mentions)
Axioplan microscope, by Leica camera (1 mentions)
Illustrator cs6, by Adobe (1 mentions)
Leica dmi6000b fluorescence microscope, by Leica Microsystems (1 mentions)
Spot inside b w qe digital camera, by Visitron (1 mentions)
Paraformaldehyde solution, by ITW Reagents (1 mentions)
A11034, by Thermo Fisher Scientific (1 mentions)
Locked nucleic acid probes, by Qiagen (1 mentions)
13 protocols using leica dfc360 fx camera
1
Evaluating Peptide-Induced Toxicity in Glioblastoma Spheroids
2
Confocal Imaging of Arabidopsis Ovules
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Confocal laser-scanning micrographs of ovules were obtained with a Zeiss 780 Inverted Axio Observer with supersensitive GaASp detector. For GFP, Venus and TdTomato detection, 488, 514, and 561 nm argon lasers were used for each respective excitation, and emissions were detected between 493 and 598 nm for GFP, between 526 and 544 nm for Venus and between 570 and 632 nm for TdTomato. Using a C-Apochromat 40× water immersion objective (numerical aperture = 1.2) confocal scans were performed with the pinhole at 1 Airy unit. Presented images show either a single focal plane or a 3D reconstruction of individual images taken as a z-series and processed using the ZEN2011 software.
Activity of the SUC2pro:GFP reporter in FG5 stage ovules was analyzed using a Leica DMI4000 inverted microscope (HXC PL fluotar 10× objective) with differential interference contrast (DIC; Nomarski) optics, a Leica DFC360FX camera, and LAS AF (Leica Microsystems) software. GFP was detected using an L5 fluorescein isothiocyanate/GFP band-pass filter.
GUS staining was analyzed using a Zeiss Axioplan microscope with DIC (Nomarski) optics, a Leica DFC295 camera and LAS core imaging software.
Adobe Illustrator CS6 and Adobe Photoshop CS6 were used to assemble photographs and to add arrows to indicate details.
Activity of the SUC2pro:GFP reporter in FG5 stage ovules was analyzed using a Leica DMI4000 inverted microscope (HXC PL fluotar 10× objective) with differential interference contrast (DIC; Nomarski) optics, a Leica DFC360FX camera, and LAS AF (Leica Microsystems) software. GFP was detected using an L5 fluorescein isothiocyanate/GFP band-pass filter.
GUS staining was analyzed using a Zeiss Axioplan microscope with DIC (Nomarski) optics, a Leica DFC295 camera and LAS core imaging software.
Adobe Illustrator CS6 and Adobe Photoshop CS6 were used to assemble photographs and to add arrows to indicate details.
3
Immunofluorescence Assay for Filovirus Nucleoproteins
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IFA was performed as described previously [49 (link)]. Viral nucleoproteins were stained using an antibody against MARV NP (mouse) and a chicken-derived antibody against EBOV NP both in combination with a species-specific Alexa Fluor® 594-conjugated or Alexa Fluor® 488-conjugated secondary antibody. DAPI (4′,6′-diamidino-2-phenylindole) staining of the nuclei was performed at a final concentration of 0.5 µg/mL. Images were acquired using a Spot inside B/W QE digital camera (Visitron Systems, Puchheim, Germany) on a Zeiss Axiophot upright fluorescence microscope (63× objective) or a LEICA DMI6000 B fluorescence microscope (63× objective, Leica Microsystems, Wetzlar, Germany) with a Leica DFC 360 FX camera (Leica Microsystems, Wetzlar, Germany).
4
Quantifying p21 Expression in MSCs
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Human adipose-derived MSCs from 4 healthy donors were seeded at a density of 104 per mL. Cells were used between passage 3 and 4. PDGF-BB was added at concentration 10 ng/mL for 24 h after 18 h of serum deprivation. MSCs were fixed with 4% paraformaldehyde solution (Panreac, Barselona, Spain) at room temperature for 10 min and incubated with 0.2% triton ×100 (Sigma) solution at RT for 10 min. Further, MSCs were incubated for 1 h in 1% bovine serum albumin (BSA, Sigma) and 10% normal goat serum (Abcam, Cambridge, UK) solution at room temperature to block the non-specific interaction of antibodies. Subsequently, the samples were incubated with primary polyclonal rabbit antibody for p21Waf1/Cip1 (Cell Signaling, 2947S) or rabbit polyclonal IgG (Biolegend, San Diego, CA, USA, 910801) in 1% BSA solution at +4° overnight. Then, samples were incubated with fluorescence-labeled goat anti-rabbit secondary antibodies (A11034, Invitrogen) at room temperature for 1 h. Cell nuclei were labeled with DAPI (DAKO). Samples were analyzed with a Leica DM6000B fluorescent microscope equipped with a Leica DFC 360FX camera (Leica Microsystems GmbH, Wetzlar, Germany). The percentage of p21-positive MSCs was evaluated.
5
Fluorescence In Situ Hybridization of Centromeric Repeats
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FISH was performed on dried preparations of nuclei from PCD samples 12 h after induction as described (Simon et al., 2018 (link)) using directly labeled Locked Nucleic Acid probes (Exiqon, Vedbæk, Denmark) specific for 180 bp centromeric repeats TEX615_GTATGATTGAGTATAAGAACTTAAACCG. Hybridization was performed for 1 h at 50°C. Post-hybridization washes were carried out at 55°C twice in 2× SSC and once in 0.75× SSC. Slides were mounted in Vectashield containing DAPI (Vector Laboratories). Imaging was performed with a Leica DMI4000 inverted microscope, a Leica DFC360FX camera, and LAS AF (Leica Microsystems) software.
6
Interphase Nuclear Spread Preparation
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Leaves at 12 h after induction of PCD were fixed in 3:1 ethanol/acetic acid and stored at 4°C. Interphase nuclear spreads for DAPI staining and FISH were prepared as previously described (Fransz et al., 2002 (link)). Briefly, leaves were rinsed in citrate buffer (10 mM Na Citrate, pH 4.5) and incubated in an enzyme mix [(0.3% (w/v) pectolyase (Sigma, St. Louis, MO, USA), 0.3% (w/v) cytohelicase (Sepracor, Marlborough, MA, USA), and 0.3% (w/v) cellulase (Sigma)] in citrate buffer for 2 h at 37°C. Digested leaves were softened in 100 µL 60% (v/v) acetic acid on a non-coated, ethanol cleaned, microscopic slide, spread at 45°C, fixed with ice-cold ethanol/acetic acid (3:1), and dried. The dried preparations were mounted in Vectashield containing DAPI (Vector Laboratories, Burlingame, CA, USA) and used for nuclear size and chromocenter number measurements. Imaging was performed with a Leica DMI4000 inverted microscope, a Leica DFC360FX camera, and LAS AF (Leica Microsystems, Wetzlar, Germany) software. Statistical significance of differences in nuclear size and number of chromocenters was tested with Wilcoxon statistical test.
7
Peptide-induced Tumor Spheroid Death
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Cell death induced by antitumor peptides on tumor cell spheroids was also analyzed by detection of PI-uptake by microscopy. After the generation of hanging drops for 72 h as described above, the peptide was carefully added at different concentrations (0–100 µM) to the hanging drops. Spheroids were then incubated in presence of peptide at 37 °C and 5% CO2 for 24 h. After the addition of 2.5 µL PI (10 µg/mL), spheroids were incubated for another 10 min before microscopic images were taken. Experiments were performed with a Leica DMI6000 B with IMC using a Leica DFC360 FX camera and AF 6000 software (Leica Microsystems, Vienna, Austria). Excitation and emission wavelengths were 536 nm and 617 nm, respectively.
8
Peptide-Induced Cell Death Visualization
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Cells (1–5 × 104) were seeded on Ibidi µ-Slide 8 wells (ibidi GmbH, Martinsried, Germany) and grown in 300 µL media for 2–3 days to a confluent layer. The peptide was added at different concentrations and incubated for 8 h at 37 °C and 5% CO2. Propidium iodide PI (2 µL/well of 50 µg/mL, Molecular Probes Inc., Eugene, OR, USA) was added to the wells and incubated for another 5 min in the dark. Experiments were performed with a Leica DMI6000 B with IMC using a Leica DFC360 FX camera and AF 6000 software (Leica Microsystems, Vienna, Austria). Excitation and emission wavelengths were 536 nm and 617 nm, respectively. PI can only enter cells and intercalate with the DNA upon potential peptide-induced membrane damage. For the visualization of the interaction of the peptide with PS and induced cell death, MUG-Mel2 cells seeded and grown for 2–3 days on Ibidi µ-Slides were incubated with 10 μM ((5-6)-FAM-) RDP22 for up to 6 h. Cells were then stained and examined with Annexin V Alexa Fluor 350 conjugate and PI as described in 2.2.2. For the visualization of the interaction of RDP22 with non-malignant control cells, NHDF were analyzed on Ibidi µ-Slides upon incubation with 10 μM ((5-6)-FAM-) RDP22 for up to 4 h and stained with Annexin V-Alexa 488 and PI as described in 2.4.
9
Visualizing Cytoskeletal Dynamics
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Non-adherent and adherent cells were separately washed with PBS and fixed in 4% formaldehyde solution for 10 minutes. Actin staining was performed using Alexa FluorTM 488 phalloidin (Invitrogen, CA, USA) according to the vendor’s instructions. The adherent cells were viewed using a Leica DMI 6000B microscope (Leica Microsystem, CA, USA) and the images were recorded using a Leica DFC 360 FX camera (Leica Microsystem, CA, USA). The 3D structures of the non-adherent colonies were visualized using a Leica TCS LSI microscope (Leica Microsystem, CA, USA).
10
Immunofluorescence Staining of MSCs
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MSCs were fixed with 4% paraformaldehyde solution (Panreac) at room temperature for 10 min and incubated with 0.2% Triton ×100 (Sigma, St. Louis, MO, USA) solution at RT for 10 min (except neurotrimin labelling). Furthermore, MSCs were incubated for 1 h in 1% bovine serum albumin (BSA, Sigma) and 10% normal goat serum (Abcam, Cambridge, UK) solution at room temperature to block the non-specific interaction of antibodies. Subsequently, the samples were incubated with primary polyclonal rabbit antibody for αSMA (Biolegend, San Diego, CA, USA, 904601), perilipin (Thermo Fisher Scientific, PA1-1051), CHD3 (Cloud-Clone Corp., Wuhan, China, PAA317Mu01), neurotrimin (Affinity Biosciences, Melbourne, Victoria, Australia, DF4245), RDH10 (Affinity Biosciences, DF12105), or rabbit polyclonal IgG (Biolegend, 910801) in 1% BSA solution at +4° overnight. Then, the samples were incubated with fluorescence-labeled goat anti-rabbit or goat anti-mouse (Invitrogen, A-11001) secondary antibodies (A11034, Invitrogen) at room temperature for 1 h. Cell nuclei were labeled with DAPI (DAKO, Glostrup, Denmark). Samples were analyzed with a Leica DM6000B fluorescent microscope equipped with a Leica DFC 360FX camera (Leica Microsystems GmbH, Wetzlar, Germany) using the LasX program. The percentage of CHD3+ MSCs was evaluated in FIJI using IgG-based thresholding.
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