Confluence and cell viability on the surface-seeded films with a total area of 6 cm2 were investigated by differential staining of living and dead cells with a Live/Dead staining kit (Invitrogen, Waltham, MA, USA). Murine fibroblasts of the 3T3 line were stained after 76 h of incubation on the surface of the films. A scanning laser confocal microscope LSM 880 Airyscan (Carl Zeiss, Oberkochen, Germany) equipped with an AiryScan module and a GaAsP detector (Carl Zeiss, Oberkochen, Germany) was used to visualize green living and red dead cells. Z scans were obtained using an EC Plan-Neofluar lens (Carl Zeiss, Oberkochen, Germany) and lasers with wavelengths of 488 and 561 nm.
Gaasp detector
Manufactured by Zeiss
Sourced in Germany
The GaAsP detector is a type of photodetector that utilizes gallium arsenide phosphide (GaAsP) as the semiconductor material. It is designed to detect and convert light signals into electrical signals, which can be used for various applications in scientific and industrial settings.
Automatically generated - may contain errors
Lab products found in correlation
Airyscan module, by Zeiss (7 mentions)
Lsm 880 airyscan, by Zeiss (2 mentions)
Alamar blue, by Thermo Fisher Scientific (2 mentions)
Picogreen, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Live dead staining kit, by Thermo Fisher Scientific (1 mentions)
Ec plan neofluar lens, by Zeiss (1 mentions)
Victor nivo, by PerkinElmer (1 mentions)
Live dead staining kit, by Merck Group (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
Syto9, by Thermo Fisher Scientific (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Airyscan, by Zeiss (1 mentions)
Plan apochromat, by Zeiss (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Zen software, by Zeiss (1 mentions)
Axio observer, by Zeiss (1 mentions)
13 protocols using gaasp detector
1
Live/Dead Cell Viability Assay on Films
2
Cytotoxicity Assessment of Biomaterials
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The cytotoxicity was studied using Live/Dead staining, AlamarBlue and PicoGreen assays. To reveal the contact cytotoxicity, the samples were seeded with MSC (1.5 × 105 cells per construct) and stained in 24 h with Live/Dead staining kit (04511, Sigma-Aldrich, USA) and Hoechst 33258 (94403, Sigma-Aldrich, USA). The prepared mounts were analyzed using a laser scanning confocal microscope LSM 880 equipped with an AiryScan module and GaAsP detector (Carl Zeiss, Germany). The elution test was carried out using AlamarBlue (DAL1025, Invitrogen, Thermo Fisher Scientific, USA) and PicoGreen assays (P11495, Invitrogen, Thermo Fisher Scientific, USA) on a plate spectrofluorimeter Victor Nivo (PerkinElmer, USA) as described elsewhere [16 , 17 (link)]. The extraction was performed using DMEM/F12 medium containing 5% FCSIII (HyClone, USA) and 1% penicillin–streptomycin (15140122, Gibco, Thermo Fisher Scientific, USA) by incubating in a CO2 incubator at a temperature of 37 °C for 24 h. Sodium dodecyl sulfate (SDS) (L3771, Sigma-Aldrich, USA) was used as a positive control.
3
Confocal Microscopy Imaging of Fluorescent Proteins
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Fluorescence imaging was performed using a Zeiss LSM800 confocal laser scanning microscope (CLSM) with a GaAsP detector (Zeiss, Germany). The manufacturer’s default settings (smart mode) were used for imaging GFP (excitation, 488 nm; emission, 495-545 nm)-, and tdTomato (excitation 561 nm; emission, 571-630 nm)-tagged proteins respectively. To image FM4-64-stained cells, a laser line of 543 nm was used for excitation, and an emission light with a wavelength of 600-700 nm was collected. For PI staining, excitation of 561 nm was used and emission signal was collected using a filter of 580- 680 nm. All images were recorded in 8 bit depth, 2 × line averaging. The images were analyzed and visualized with Fiji program [76 (link)].
4
Confocal Microscopy Analysis of Biofilm Extracellular Matrix
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Biofilms were analyzed at 43 h via confocal microscopy. All procedures
for biofilm formation and topical treatments were as described above,
except that 13.4 μL of 1 mM Alexa Fluor 647 fluorophore-labeled
dextrans (647/668 nm; Molecular Probes, Carlsbad, CA, EUA) was added
to the culture medium to label the extracellular matrix (at 0, 19,
and 27 h). At 43 h, the biofilms were dip-washed on a 24-well plate
containing 0.89% NaCl and incubated in another 24-well plate containing
0.89% NaCl with 1.5 μL of SYTO9 (485/498 nm; Molecular Probes)
(30 min) to label microorganisms.5 (link),40 (link) After, they
were dip-washed on a new 24-well plate with 0.89% NaCl and imaged.
A confocal microscope (Carl Zeiss LSM 800 with Airyscan and a GaAsp
detector, Germany) was used with an EC Plan-Neofluar 20×/0.50
Oil DIC M27 objective, with laser wavelengths (488 nm, 2.10%; and
561 nm, 1.81%), with increments of 1.5 μm. The images were analyzed
using ZEN Blue software to quantify the biomass, maximum thickness,
and percentage of coverage area using COMSTAT2.
Three experimental
occasions were performed. Two discs represented
each treatment group (formulation), and three images were acquired
per disc avoiding the disc’s edges (n = 6).
All data files were used for the quantification analyses, and a representative
image from each group was selected to illustrate the findings.
for biofilm formation and topical treatments were as described above,
except that 13.4 μL of 1 mM Alexa Fluor 647 fluorophore-labeled
dextrans (647/668 nm; Molecular Probes, Carlsbad, CA, EUA) was added
to the culture medium to label the extracellular matrix (at 0, 19,
and 27 h). At 43 h, the biofilms were dip-washed on a 24-well plate
containing 0.89% NaCl and incubated in another 24-well plate containing
0.89% NaCl with 1.5 μL of SYTO9 (485/498 nm; Molecular Probes)
(30 min) to label microorganisms.5 (link),40 (link) After, they
were dip-washed on a new 24-well plate with 0.89% NaCl and imaged.
A confocal microscope (Carl Zeiss LSM 800 with Airyscan and a GaAsp
detector, Germany) was used with an EC Plan-Neofluar 20×/0.50
Oil DIC M27 objective, with laser wavelengths (488 nm, 2.10%; and
561 nm, 1.81%), with increments of 1.5 μm. The images were analyzed
using ZEN Blue software to quantify the biomass, maximum thickness,
and percentage of coverage area using COMSTAT2.
Three experimental
occasions were performed. Two discs represented
each treatment group (formulation), and three images were acquired
per disc avoiding the disc’s edges (n = 6).
All data files were used for the quantification analyses, and a representative
image from each group was selected to illustrate the findings.
5
Fibrinogen Fluorescent Labeling and Imaging
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The procedures were performed, as described elsewhere.57 (link),58 Briefly, before polymerization, fibrinogen solutions were mixed with fibrinogen conjugated with AlexaFluor-488 (Invitrogen, USA) at a ratio 50:1. Samples were prepared on slides and analyzed using a confocal laser scanning microscope LSM 880 equipped with an AiryScan module and GaAsP detector (Carl Zeiss, Germany; water immersion objective).
6
Fluorescent Imaging of Nuclei and Cytoskeleton
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The nuclei in the prepared AFM samples were stained with Hoechst 33342 dye (2 µg/mL) to detect enucleated cells or to distinguish the nucleoplasts and isolated nuclei from the cell debris. Phase-contrast and fluorescent images were recorded with 10x/0.3 or 20x/0.4 objectives using the ZEN software (Carl Zeiss, Germany) of the Axio Observer inverted fluorescent microscope.
For the confocal microscopy studies, the samples were fixed in a 4% formaldehyde solution in PBS for 10 min, permeabilized with 0.1% Triton X-100 for 10 min, blocked with 1% bovine serum albumin for 10 min and stained with Alexa Fluor 488 phalloidin (Life Technologies, USA). The samples were washed with PBS and mounted with the ProLong Gold antifade reagent (Invitrogen, USA). Fluorescent images (Z-stacks) were acquired using an LSM 880 confocal laser scanning microscope equipped with an AiryScan module and a GaAsP detector (Carl Zeiss, Germany) with a Plan-Apochromat 63x/1.4 N.A. oil immersion objective.
For the confocal microscopy studies, the samples were fixed in a 4% formaldehyde solution in PBS for 10 min, permeabilized with 0.1% Triton X-100 for 10 min, blocked with 1% bovine serum albumin for 10 min and stained with Alexa Fluor 488 phalloidin (Life Technologies, USA). The samples were washed with PBS and mounted with the ProLong Gold antifade reagent (Invitrogen, USA). Fluorescent images (Z-stacks) were acquired using an LSM 880 confocal laser scanning microscope equipped with an AiryScan module and a GaAsP detector (Carl Zeiss, Germany) with a Plan-Apochromat 63x/1.4 N.A. oil immersion objective.
7
Otic Capsule Cochlea Preparation
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Mice were euthanized based on American Veterinary Medical Association (AVMA) guidelines appropriately for the age of the animal immediately before imaging. Prehearing animals were placed on a heating pad to maintain core body temperature before euthanasia. The otic capsule preparation was adapted from the in situ cochlea preparation described in Sirko and colleagues, wherein the hemidissected head was placed in 2.5 mm K+ external solution on ice and the otic capsule excised from the temporal bone (Sirko et al., 2019 (link)). Then, the bone overlying the cochlear epithelium was removed. The fenestrated otic capsule cochlea was mounted on utility wax for observation at room temperature. A gravity perfusion system was constructed on a motorized stage of a 710 LSM Zeiss microscope with a GaAsP detector (Zeiss) and Chameleon two-photon laser (Coherent). The preparation was placed in external solution and imaged using a 20× non-coverslip-corrected water-immersion objective (Nikon). With an objective in place, the two-photon laser measured 299 mW at 100% power when set to the 920-nm wavelength used in these experiments.
8
Cytotoxicity Assessment of Biomaterials
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The material cytotoxicity was assessed using Live/Dead (Sigma Aldrich, Steinheim am Albuch, Germany) staining and AlamarBlue (Invitrogen, Carlsbad, CA, USA) and PicoGreen (Invitrogen, Carlsbad, CA, USA) assays. We used mesenchymal stromal cells (MSC) derived from the gingiva. The cells were cultured as described elsewhere [29 (link)] and monitored using a phase contrast microscope. A scaffold was inoculated with 50,000 cells and stained with calcein-AM (green, live cells) and propidium iodide (red, dead cells) in 5 days. Then it was analyzed using an LSM 880 laser scanning confocal microscope equipped with an AiryScan module and GaAsP detector (Carl Zeiss, Jena, Germany). For colorimetric assays, the material extract was prepared as previously described [30 ] and used to prepare serial dilutions. The sample solution or sodium dodecyl sulfate (SDS, positive control) were added to the cells cultured in a 96-well plate for 24 h at a temperature of 37 °C.
9
Quantifying Fibrin Porosity via Confocal Microscopy
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The procedures were performed as described elsewhere.35–37 (link) Briefly, before polymerization, fibrinogen solutions were mixed with fibrinogen conjugated with AlexaFluor-488 (Invitrogen, USA) at a ratio 50 : 1. Samples were prepared on slides and analyzed using an LSM 880 confocal laser scanning microscope equipped with an AiryScan module and GaAsP detector (Carl Zeiss, Germany; 40× water immersion objective). Porosity was measured in ten images from three samples using the ImageJ software (NIH, USA).
10
Immunocytochemistry of LUHMES Neuronal Cells
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LUHMES cells, cultured and differentiated on pre-coated glass bottom 8-well µ-slides
(Ibidi, Germany) at cell density of 150 000 cells/cm2, were fixed with 4%
paraformaldehyde (Sigma Aldrich) for 15 min at RT, washed and permeabilized with 0.2%
Triton X-100 in phosphate buffered saline (PBS) for 10 min at RT. Blocking solution of 5%
bovine serum albumin (Calbiochem, USA) was then added for 1 h at RT. Mouse anti-TUJ1
primary antibody (Covance, USA) diluted 1:500 was then added overnight at 4 °C. Samples
were washed three times with PBS/0.05% Tween and anti-mouse Alexa-488 secondary antibody
(Invitrogen) were applied for 1 h at RT in dark. 1 µg/ml Hoechst-33342 (Molecular Probes,
USA) was added 10 min before the incubation with the secondary antibody was over. Cells
were then washed three times with PBS and imaged with LSM 880 confocal point laser
scanning microscope equipped with a GaAsP detector (Zeiss, Germany) using a 40× oil
objective. Image processing was carried out with the Fiji software.
(Ibidi, Germany) at cell density of 150 000 cells/cm2, were fixed with 4%
paraformaldehyde (Sigma Aldrich) for 15 min at RT, washed and permeabilized with 0.2%
Triton X-100 in phosphate buffered saline (PBS) for 10 min at RT. Blocking solution of 5%
bovine serum albumin (Calbiochem, USA) was then added for 1 h at RT. Mouse anti-TUJ1
primary antibody (Covance, USA) diluted 1:500 was then added overnight at 4 °C. Samples
were washed three times with PBS/0.05% Tween and anti-mouse Alexa-488 secondary antibody
(Invitrogen) were applied for 1 h at RT in dark. 1 µg/ml Hoechst-33342 (Molecular Probes,
USA) was added 10 min before the incubation with the secondary antibody was over. Cells
were then washed three times with PBS and imaged with LSM 880 confocal point laser
scanning microscope equipped with a GaAsP detector (Zeiss, Germany) using a 40× oil
objective. Image processing was carried out with the Fiji software.
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