Live-cell imaging of cells expressing GFP-EBP50 (Garbett and Bretscher, 2012 (link)) was done using an inverted Leica DMi8 widefield microscope equipped with a Leica ×100 NA air objective, a Leica DFC 9000 GTC camera, Leica Application Suite X THUNDER deconvolution software, and Leica adaptive focus control. Jeg3 cells were transiently transfected 24 hr prior to imaging, and the cell medium was changed to FluoroBrite DMEM (Thermo Fisher Scientific; Cat# A1896701) with 10% FBS immediately before imaging. The cells were then kept in a 5% CO2 humidified environmental chamber at 37°C throughout the duration of the imaging as cells were imaged every 1 min for 15 min. Deconvolution images of GFP-EBP50 were performed on the Leica DMi8 widefield microscope, Leica Application Suite X THUNDER deconvolution software, and Leica adaptive focus control listed above. The default small sample Leica THUNDER deconvolution settings were used for this, apart from adjusting structure size to 1000 nm and reducing deconvolution strength to 30%.
Dmi8 widefield microscope
Manufactured by Leica
Sourced in United Kingdom
The Leica DMi8 is a widefield microscope designed for observation and imaging of samples. It features a modular design, allowing for customization to meet specific research requirements. The DMi8 provides high-quality optics and illumination, enabling detailed observation and analysis of specimens.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Las x software, by Leica (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Chabc, by Merck Group (2 mentions)
Aggrecan, by Merck Group (2 mentions)
Dfc9000 gtc camera, by Leica (2 mentions)
Las x premium software, by Leica (2 mentions)
Dfc9000gt, by Leica (2 mentions)
Application suite x, by Leica (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Neutral buffered formalin, by Merck Group (1 mentions)
Euthasol, by Virbac (1 mentions)
Amg evos fl digital inverted microscope, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit alexa fluor 555, by Abcam (1 mentions)
Alexa fluor 488 goat anti chicken, by Abcam (1 mentions)
Alexa fluor 555 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Torpedodna, by Ibidi (1 mentions)
39 protocols using dmi8 widefield microscope
1
Live-cell Imaging of GFP-EBP50 Expressing Cells
2
Quantifying Cardiac Fibrosis in Mice
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Hearts were excised from anesthetized mice (Euthasol, 200 mg/kg i.p., Virbac AH, Inc., Fort Worth, TX, USA), immediately fixed in 10% neutral buffered formalin (Sigma, Saint Louis, MO, USA) and processed as previously described [15 (link)]. Briefly, for histology studies, paraffin-embedded hearts were sectioned (4 μm), deparaffinized, rehydrated, incubated in 0.1% Sirius red in saturated picric acid for 1 h and then washed in acidified water. Paraffin-embedment and slide preparation were carried out by the University of Cincinnati Pathology Department Core Facility. Images were acquired on a Leica DMi8 Widefield microscope with a 20× objective lens using LASX software (Leica Microsystems, 9435 Heerbrugg, Switzerland). The analysis of fibrosis levels in cross sections of LV and RV was performed by calculating the collagen fiber staining with Image J (version 1.48v; National Institutes of Health, Bethesda, Maryland, MD, USA).
3
Immunofluorescence Staining Protocol
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Cells were fixed in 4% paraformaldehyde (PFA) for 15 min at room temperature (RT) and permeabilized with 0.1% Triton X-100 for 15 min at RT. Fixed cells were blocked with 2% bovine serum albumin (BSA) (Sigma-Aldrich) for 4 h at RT and then incubated overnight with the primary antibodies (Table 1 ) at 4°C. The following day, cells were washed thrice with dPBS and blocked again for 1 hour with 2% BSA at RT. Secondary antibodies, such as goat anti-mouse Alexa Fluor 488 (Thermo Fisher Scientific), goat anti-rabbit Alexa Fluor 555 (Abcam), mouse anti-goat Alexa Fluor 555 (Abcam), goat anti-chicken Alexa Fluor 488 (Abcam), and goat anti-mouse Alexa Fluor 555 (Thermo Fisher Scientific), at 1:500 were added for 1 hour at RT. Cells were triple-washed with dPBS, and nuclei were stained using DAPI (Invitrogen) at 1:10,000 diluted in a 1x dPBS solution for 5 min. Cells were washed once after the DAPI addition with dPBS and mounted using the ProLong Gold Antifade Reagent (Invitrogen). Images were acquired using the AMG EVOS FL digital inverted microscope (Thermo Fisher Scientific.) and the LEICA DMi8 wide-field microscope (Leica Microsystems).
4
Live Cell Imaging of Transfected Cos7 Cells
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Cos7 cells were seeded on 8w µ-slides (ibidi) at a density of 2–4 × 104 cells per cm2 and transiently transfected with plasmid DNA using TorpedoDNA (ibidi) on the next day according to the manufacturer’s instructions. Then, 24 h post-transfection cells were stained with 1 µg ml−1 Hoechst 33342 (Thermo Fisher Scientific) in PBS for 10 min to label nuclei. After staining, cells were washed three times with PBS (Sigma Aldrich) and supplemented with phenol red-free growth medium (DMEM, Sigma Aldrich) for live cell imaging. Images were acquired with a Leica DMi8 wide-field microscope (Leica microsystems) using a 100x magnification objective and the manufacturer’s LAS X 2 software. Filters: GFP (Ex.: 450–490, Em.: 500–550), DAPI (Ex.: 325–375, Em.: 435–485). Greyscale images were transformed into RGB color images and RGB merged in green or blue, respectively, with ImageJ 1.37a. RGB images were overlaid with Photoshop Version 11.0 without any further adjustments.
5
Quantification of Azide-LA Binding in S. aureus
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S. aureus USA300 JE2 WT, ΔessC, and ΔesxC, grown to OD600 of 1.0, were treated with 10 µM azide-LA for 15 min at 37 °C with shaking. The samples were then centrifuged, and the bacterial pellets resuspended in PBS supplemented with 4 µg/mL Click-iT Alexa Fluor 488 sDIBO alkyne (Life Technologies LTD, UK). After incubation at 25 °C for 1 h with shaking, bacteria were washed with PBS, and binding to azide-LA was quantified by measuring fluorescence using a FLUOstar OMEGA plate reader (BMG Labtech, UK). The samples imaged with a microscope were additionally stained with 3 µM propidium iodide, following click chemistry. Bacteria stained with Click-iT Alexa Fluor 488 sDIBO alkyne and 3 µM propidium iodide were immobilized on agarose-covered glass slides, and viewed with a Leica DMi8 widefield microscope (Leica Microsystems LTD, UK). Images were analysed with the ImageJ processing package Fiji61 (link).
6
Fluorescent Labeling of MSC-Derived Exosomes
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MSC-Exos were labeled using the PKH67 Green Fluorescent Cell Linker Kit for
General Cell Membrane Labeling (Sigma-Aldrich) according to the manufacturer’s
instructions. After preparation, samples were then centrifuged at 190,000 g for
2 hours at 4 °C. Following ultracentrifugation, the medium and interface layer
was aspirated out and the fluorescently labeled MSC-Exo pellet was resuspended
in sterile 1X PBS.
Bovine AF cells (p2) were plated in a Corning® Costar® 6-well Clear TC-Treated
Multiple Well Plate (Corning Inc.) at a seeding density of 4000
cells/cm2 in complete growth medium (2 mL/well). When 65%
confluence was reached, complete growth medium was fully exchanged with
serum-free medium for the following 2 conditions: (1) untreated serum-free
medium and (2) serum-free medium supplemented with the total resuspended volume
of fluorescently labeled MSC-Exos. Plates were incubated at 37 °C, 5%
CO2 for 6 hours. Following the 6-hour incubation period, AF cells
were fixed with ice cold 4% PFA (Electron Microscopy Sciences) for 15 minutes,
washed twice with 1X PBS (5 min/wash), and immediately imaged on a Leica DMi8
widefield microscope (Leica Microsystems Inc.).
General Cell Membrane Labeling (Sigma-Aldrich) according to the manufacturer’s
instructions. After preparation, samples were then centrifuged at 190,000 g for
2 hours at 4 °C. Following ultracentrifugation, the medium and interface layer
was aspirated out and the fluorescently labeled MSC-Exo pellet was resuspended
in sterile 1X PBS.
Bovine AF cells (p2) were plated in a Corning® Costar® 6-well Clear TC-Treated
Multiple Well Plate (Corning Inc.) at a seeding density of 4000
cells/cm2 in complete growth medium (2 mL/well). When 65%
confluence was reached, complete growth medium was fully exchanged with
serum-free medium for the following 2 conditions: (1) untreated serum-free
medium and (2) serum-free medium supplemented with the total resuspended volume
of fluorescently labeled MSC-Exos. Plates were incubated at 37 °C, 5%
CO2 for 6 hours. Following the 6-hour incubation period, AF cells
were fixed with ice cold 4% PFA (Electron Microscopy Sciences) for 15 minutes,
washed twice with 1X PBS (5 min/wash), and immediately imaged on a Leica DMi8
widefield microscope (Leica Microsystems Inc.).
7
Quantifying Lipid Peroxidation by BODIPY-C11 Imaging
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Lipid peroxidation was detected and quantified using BODIPY-C11 probe by fluorescent microscopy. Images were quantified using ImageJ Fiji. Cells were imaged using a fully motorized Leica DMi8 widefield microscope (from Leica Microsystems) using the fluorescein isothiocyanate and Texas Red filter sets, and a ×20 objective. All imaging acquisition parameters were kept constant for each experiment. Images were quantified using ImageJ Fiji. Cell outlines were free-handed drawn on the bright-field channel to generate a cell selection mask for quantifying the fluorescence intensity in the green and red channels. Oxidation of BODIPY-C11 581/591 was calculated as the ratio of the green (fluorescence emission of the oxidized probe)/red fluorescence mean intensity (fluorescence emission of reduced probe) within the cell outlines. Imaging was performed on two independent biological replicates. In each independent experiment at least 4 different images (100 cells) per condition were analyzed. Each experiment was performed in duplicate. At least three independent experiments were performed for each cell type and condition.
8
Linoleic Acid-Induced Bacterial Viability
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Bacteria grown to OD600 of 1.0, were treated with 80 μM linoleic acid for 15 min at 37 °C with shaking. The samples were then centrifuged, and the bacterial pellets resuspended in PBS and supplemented with a 1:1 ratio of 2X LIVE/DEAD solution (6 μM SYTO-9 stain and 30 μM propidium iodide) from LIVE/DEAD BacLight kit (Invitrogen). After incubation in the dark for 15 min, bacteria were washed with PBS, spotted on to agarose pads and imaged using a Leica DMi8 widefield microscope (Leica Microsystems, UK). Acquired images were analysed with the ImageJ processing package, Fiji.
9
Neural Stem Cell Migration on CSPG Substrates
10
Neural Stem Cell Migration on CSPG Substrates
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To determine the effects of CSPGs on the migration of neural stem
cells in vitro, flat-bottom 48-well plates were coated with
poly-L-lysine and various concentrations (1ug/mL and 10ug/mL) of Aggrecan
(A1960, sigma). The control wells contained poly-L-lysine alone.
Neurospheres of similar size were seeded in each well in NBM-GF medium with
2.5μM ISP peptide or scrambled peptide (n = 7 neurospheres per
condition), and the plates were incubated at 37°C for 21 hours.
Thereafter, images of each well were taken using a Leica DMi8 widefield
microscope. The Migration Index was defined as dividing the total area of
migrated cells by the inner area of neurospheres. The inner area and total
area of neurospheres were measured using ImageJ software. Time lapse
analyses (duration 21 hours) of the migration process (Video S1 , Supplementary data ) was taken
using a Leica DMi8 widefield microscope equipped with an on-stage incubator.
For application of signaling pathway inhibitors, neurospheres were treated
with LY294002 (10μM, AKT inhibition), PD98059 (10μM, ERK
inhibition) or OA-Hy (100nM, MMP2 inhibition) for 30min followed by exposure
to 2.5μM ISP peptide or scrambled peptide. For ChABC treatment,
neurospheres were incubated with 5 mU/mL ChABC (Sigma, # C2905) for 21 hours
and then NSCs migration was measured. Each experiment was repeated twice
with similar results and representative results are presented.
cells in vitro, flat-bottom 48-well plates were coated with
poly-L-lysine and various concentrations (1ug/mL and 10ug/mL) of Aggrecan
(A1960, sigma). The control wells contained poly-L-lysine alone.
Neurospheres of similar size were seeded in each well in NBM-GF medium with
2.5μM ISP peptide or scrambled peptide (n = 7 neurospheres per
condition), and the plates were incubated at 37°C for 21 hours.
Thereafter, images of each well were taken using a Leica DMi8 widefield
microscope. The Migration Index was defined as dividing the total area of
migrated cells by the inner area of neurospheres. The inner area and total
area of neurospheres were measured using ImageJ software. Time lapse
analyses (duration 21 hours) of the migration process (
using a Leica DMi8 widefield microscope equipped with an on-stage incubator.
For application of signaling pathway inhibitors, neurospheres were treated
with LY294002 (10μM, AKT inhibition), PD98059 (10μM, ERK
inhibition) or OA-Hy (100nM, MMP2 inhibition) for 30min followed by exposure
to 2.5μM ISP peptide or scrambled peptide. For ChABC treatment,
neurospheres were incubated with 5 mU/mL ChABC (Sigma, # C2905) for 21 hours
and then NSCs migration was measured. Each experiment was repeated twice
with similar results and representative results are presented.
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