The crayfish did not show any melanization before the experiments. All crayfish were checked upon arrival to the laboratory and before being challenged with zoospores. Crayfish were checked daily for disease symptoms and dead crayfish were removed and examined for the presence of melanized areas both macroscopically and microscopically. For microscopic examination, the subabdominal cuticle was removed and observed using an inverted microscope Olympus CKX41SF (Olympus Optical, Tokyo, Japan). Light micrographs were captured using a Qimaging Micropublisher 5.0 digital camera (Qimaging, Burnaby, BC, Canada). Digital image analysis was performed using the software Syncroscopy-Automontage (Microbiology International Inc., Frederick, MD) as described in Diéguez-Uribeondo et al. 2003 [28 ]. After 120 days of the challenge experiments, live crayfish were euthanized [26 (link)] and examined as described above.
Ckx41sf
Manufactured by Olympus
Sourced in Japan, Germany, United States
The CKX41SF is a compact inverted tissue culture microscope designed for routine cell culture observation. It features a monochrome LED illumination system and a tilting binocular viewing head with a wide field of view. The microscope is suitable for a variety of cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
L glutamine, by Merck Group (5 mentions)
Ascorbic acid 2 phosphate, by Merck Group (4 mentions)
α mem, by Thermo Fisher Scientific (4 mentions)
Penicillin, by Merck Group (4 mentions)
Streptomycin, by Merck Group (4 mentions)
Matrigel, by Merck Group (2 mentions)
Cetylpyridinium chloride, by Merck Group (2 mentions)
Alizarin red s solution, by ScienCell (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Cellular senescence assay, by Merck Group (1 mentions)
Trypan blue staining, by Merck Group (1 mentions)
Pipette tip, by Eppendorf (1 mentions)
Trypsin edta, by GE Healthcare (1 mentions)
Q capture software, by Olympus (1 mentions)
Q color 3, by Olympus (1 mentions)
Methocult m3630, by STEMCELL (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Transwell plate, by Corning (1 mentions)
78 protocols using ckx41sf
1
Melanization Assessment in Challenged Crayfish
2
Microscopic Examination of Crayfish Pathogen
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All crayfish were examined before the start of the experiments to ensure they did not show any signs of previous colonization by the pathogen and resistance by checking for the presence of melanized hyphae. After being challenged with zoospores, crayfish were checked daily for disease symptoms. Dead crayfish were removed and examined, both macroscopically and microscopically, for the presence of melanized areas. For the microscopic examination, the sub-abdominal cuticle was removed and observed on an inverted microscope Olympus CKX41SF (Olympus Optical, Tokyo, Japan). Light micrographs were captured using a Qimaging Micropublisher 5.0 digital camera (Qimaging, Burnaby, BC, Canada). Digital image analysis was performed using the software Syncroscopy-Automontage (Microbiology International Inc., Frederick, MD, USA.) as described by Diéguez-Uribeondo et al. [51 (link)].
3
Adenovirus-Mediated Nrf2 Overexpression in A549 Cells
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A549 cells were grown in a monolayer culture in RPMI-1640 medium supplemented with 10% FBS at 37°C in a humidified atmosphere with 5% CO2. Following this incubation, cells (4×104 cells/well) were inoculated onto a 96-well culture plate and left to grow in the logarithmic stage for 24 h at 37°C. A549 cells were transfected with adenoviral vectors containing either AD-Nrf2 or AD-(GFP) (cat. no. PEP033; Thermo Fisher Scientific, Inc.), at a multiplicity of infection of 50. Control cells were cultured in RPMI-1640 medium only. The cells were cultured in RPMI-1640 medium supplemented with 10% FBS and maintained at 37°C in a 5% CO2-humidified incubator for 24 h. A fluorescence microscope (magnification, ×100; Olympus CKX41SF; Olympus Corporation, Tokyo, Japan) was used to determine the percentage of GFP synthesizing cells. The ratio of cells that emitted green fluorescence in the same field of view was regarded as the transfection efficiency. Nrf2 overexpression was also confirmed via western blotting.
4
Quantification of Cellular Senescence by SA-β-Gal Assay
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To quantify the proportion of senescence associated β-galactosidase positive (SA-β-gal+) cells, the commercial kit “Cellular Senescence Assay” (EMD Millipore, Burlington, VT, USA, Catalog Number: KAA002) was used. The cells were stained according to supplemented manufacturer protocol with the following modification: at the final PBS washing step, the cell nuclei were stained with 1 μg/mL Hoechst 33342 (Molecular Probes, Eugene, OR, USA). Such modification significantly improves the quality of counting of β-galactosidase negative cells [24 (link),25 (link)]. The stained cells were visualized using Excitation/Emission Interference Filters (CKX-U: 340–380 nm/435–485 nm) on the inverted fluorescent microscope Olympus CKX 41 SF (Olympus, Tokyo, Japan) equipped with Infinity 3-1 (Lumenera Copr., Ottawa, Canada) camera and 20× objective. The proportions of SA-β-gal+ cells were counted manually (300 cells per each data point).
5
Lentiviral Transfection of NP Cells
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To quantify the percentage of successfully transfected NP cells at a given multiplicity of infection (MOI), an identical procedure was performed with the LV-green fluorescent protein (GFP) (Shanghai Genechem Co., Ltd.) for each MOI assessed. NP cells from passage 2 were plated as a monolayer in 96-well plates at 4×104 cells/ml and were incubated for 24 h. Solutions of the viral particles equal to 30, 40, 60, 80 and 100 MOI were pre-mixed with DMEM/F12 medium and were added to the 96-well plates. After 48 h, the NP cells were examined under a fluorescence microscope (Olympus CKX41SF; Olympus Corp.), and the percentage of NP cells synthesizing GFP was determined.
NP cells from passage 2 were divided into 3 groups (the positive, negative control and blank control groups), which were transfected with LV with survivin, the empty LV or an equal amount of DMEM/F12 medium, respectively. The transfection procedure was performed with an MOI value of 50. The transfected NP cells were incubated in a CO2 incubator at 37°C. After 8 h, the growth medium was changed.
NP cells from passage 2 were divided into 3 groups (the positive, negative control and blank control groups), which were transfected with LV with survivin, the empty LV or an equal amount of DMEM/F12 medium, respectively. The transfection procedure was performed with an MOI value of 50. The transfected NP cells were incubated in a CO2 incubator at 37°C. After 8 h, the growth medium was changed.
6
Quantification of Primary Osteoblasts
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The discs with adherent human primary osteoblasts were washed with 1x PBS, treated with 200 μl of 1x Trypsin/EDTA (PAA Laboratories GmbH, Cölbe, Germany) for 3 minutes under aerobic conditions at 37°C and 5% CO2. Finally, the cells were mechanically removed from the discs with a pipette tip (Eppendorf AG, Hamburg, Germany). The solution was transferred to a 1.5-ml Eppendorf reaction tube, centrifuged at 900 rpm for 4 minutes at 4°C, and washed with 1x PBS. Quantification of living primary osteoblasts on the test samples was performed by trypan-blue staining (Sigma-Aldrich), and subsequent counting of living cells using an Abbe-Zeiss counting cell chamber (Carl Zeiss AG, Jena, Germany) under a light optical microscope (Olympus CKX41SF, Olympus GmbH, Hamburg, Germany).
7
Quantifying Pre-B Cell Colony Formation
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Bone marrow cells of Lgals3+/+ and Lgals3−/− mice were adjusted to 5.0 × 105 cells in RPMI-1640 (Sigma-Aldrich, USA) supplemented with 10% Fetal Bovine Serum, 2 mM glutamine, 10−5 β-mercaptoethanol, and 100 mg/mL penicillin and streptomycin and maintained in 25 cm2 tissue culture flasks at 37 °C in 5% CO2 atmosphere. After 1 week in culture, adherent bone marrow cells were submitted to RNA extraction protocol as previously described41 (link). To methylcellulose medium enriched with IL-7 (Methocult M3630, Stem Cell Technologies, Canada), 5 × 104 total bone marrow cells were homogenously distributed in the flasks and maintained at 37 °C in 5% CO2 atmosphere for 1 week. The colonies (more than 50 cells) and clusters (less than 50 cells) of pre-B cells were quantified through inverted microscope Olympus CKX41SF (Olympus, Japan). The capture of images was performed using camera QColor-3 attached to the Q-Capture software (Olympus).
8
In Vitro Blood-Brain Barrier Model
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Neurons were plated at the bottom of Transwell plates (0.4 μm, Corning, New York, NY, USA) and maintained in Neurobasal-A medium containing 2% B27, 0.25% GlutaMAX and 1% penicillin/streptomycin for at least 48 hours. Astrocytes (3 × 105 cells/cm2) were plated under the insert membrane in a petri dish at 44 hours. After 4 hours, the transwell inserts were inverted and transferred to the plates. At 52 hours, BMECs were plated at a density of 2 × 105 cells/cm2 in 0.5 mL of medium on the inside of the gelatin-coated insert. The experiments were initiated 120 hours after establishing the NVU cultures (at 172 hours). The procedure for establishing the in vitro NVU model is schematized in Figure 1
9
Osteogenic Differentiation of Gingiva-Derived Mesenchymal Stem Cells
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Following the previously described procedures, gingiva-derived mesenchymal stem cells (GMSCs) were obtained [14 (link)]. The tissues from the gingiva were de-epithelialized, diced, and enzyme-digested. The culture media was changed every two to three days, and the stem cells were put into a culture dish.
Stem cells were seeded at a density of 1 × 106 cells/well on silicon elastomer-based concave microwells (StemFIT 3D; MicroFIT, Seongnam-si, Republic of Korea) and grown in an osteogenic medium. A commercially available enamel matrix derivative (Emdogain®, Straumann, Basel, Switzerland) was diluted with a final concentration of 2.7, 27, 270, and 2700 μg/mL. Every one to two days, a new medium was used in place of the old one. On days 1, 3, 5, and 7, morphological analysis was performed using an inverted microscope (CKX41SF, Olympus Corporation, Tokyo, Japan).
Stem cells were seeded at a density of 1 × 106 cells/well on silicon elastomer-based concave microwells (StemFIT 3D; MicroFIT, Seongnam-si, Republic of Korea) and grown in an osteogenic medium. A commercially available enamel matrix derivative (Emdogain®, Straumann, Basel, Switzerland) was diluted with a final concentration of 2.7, 27, 270, and 2700 μg/mL. Every one to two days, a new medium was used in place of the old one. On days 1, 3, 5, and 7, morphological analysis was performed using an inverted microscope (CKX41SF, Olympus Corporation, Tokyo, Japan).
10
Quantitative Assessment of Axonal Degeneration
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Sciatic nerves were harvested from three rats per group anesthetized with sevoflurane (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) on day 30. Sciatic nerves were embedded in Epon after fixing in glutaraldehyde (2%), followed by sucrose (8%) substitution. The slice samples were stained with toluidine blue18 (link). Each section was evaluated using light microscopy (CKX41SF; Olympus Co.). The axon circularity was calculated as a quantitative index of axonal degeneration37 (link). The quantitative data were acquired from 4,061–4,919 fibers of three animals per group.
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