For wound healing and cell migration assays, HaCaT and Fibroblast cells were seeded in 24-well plates at 0.5 × 106 cells/ml. Twenty-four hours later, the medium was changed to non-FBS-containing media. The following day confluent cells were uniformly scraped with a 200-μl pipette tip across the well. Following wounding, culture medium was washed with PBS and replaced with fresh serum-free medium, and cells were continually exposed to EVs for the indicated time periods. The scratched region was photographed immediately and every 12 h after scratching using an EVOS M5000 microscope (Invitrogen) at × 20 magnification. Total scratch area was analyzed using ImageJ.
Evos m5000 microscope
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, China
The EVOS M5000 is a high-performance fluorescence microscope designed for a wide range of cell imaging applications. It features a 12 MP camera, LED illumination, and an automated stage for capturing high-quality images and time-lapse videos. The EVOS M5000 provides users with a versatile and user-friendly platform for their research needs.
Automatically generated - may contain errors
Lab products found in correlation
Accuri c6 cytometer, by BD (5 mentions)
Rnaimax, by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Opti mem, by Thermo Fisher Scientific (4 mentions)
Propidium iodide, by Thermo Fisher Scientific (2 mentions)
Hla a, by Abcam (2 mentions)
Hla dr, by Abcam (2 mentions)
Alexa fluor 488 donkey anti rabbit conjugated secondary antibodies, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 donkey anti mouse conjugated secondary antibodies, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Mitotracker green fm, by Thermo Fisher Scientific (2 mentions)
H2dcfda, by Thermo Fisher Scientific (2 mentions)
Live dead cell imaging kit, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Merck Group (2 mentions)
Genex plus reagent, by American Type Culture Collection (2 mentions)
Doxorubicin, by Cayman Chemical (2 mentions)
Dmem with pyruvate, by Thermo Fisher Scientific (2 mentions)
Rpe 1 htert cells, by American Type Culture Collection (2 mentions)
Lookout detection kit, by Merck Group (2 mentions)
Actinomycin d, by Cayman Chemical (2 mentions)
98 protocols using evos m5000 microscope
1
Wound Healing and Cell Migration Assays
2
Phenotypic Characterization of Engineered Cell Sheets
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The engineered cell sheets were fixed in 10% neutral buffered formalin and embedded in paraffin. The tissue sections were used for immunofluorescent staining with CD19 (Cat # NBP2-25196), CD73 (Cat # NBP2-25237) (NovusBIo, CO, United States), CD29 (Cat # ab134179), HLA-A (Cat # ab52922), HLA-DR (Cat # ab92511) (Abcam, MA, United States), and Oct3/4 (Cat # NB-100-2379SS) (Novus Biologicals LLC., Littleton, CO, United States). Alexa Fluor 488 donkey anti-rabbit conjugated secondary antibodies and Alexa Fluor 488 donkey anti-mouse conjugated secondary antibodies (Invitrogen, Carlsbad, CA, United States) were used. Propidium iodide (Invitrogen, Carlsbad, CA, United States) was used to stain nuclear DNA. An EVOS M5000 microscope was used to analyze the slides (Invitrogen, Carlsbad, CA, United States).
3
Quantifying Palbociclib-Induced Apoptosis
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To determine caspase-dependent apoptosis after treatment with Palbociclib, 2000 cells per well were seeded and grown overnight in 96-well plates. Triplicates were treated with increasing concentrations of the inhibitors for indicated amount of time. Caspase-Glo 3/7 (Promega, #G8091) and Cell Titer-Blue (Promega, #G8081) assays were conducted in parallel according to the manufacturer’s instructions. The caspase 3/7 activity from the Caspase-Glo assay was normalized to the number of cells present in each condition as determined by Cell Titer-Blue assay. In parallel, 2 × 106 were seeded in 10-cm plates overnight and treated with increasing concentrations of the inhibitors. Medium was aspirated and cells were harvested and stained with Trypan Blue. Stained cells were visualized and quantified using Invitrogen EVOS M5000 microscope and counting ×10 optical fields.
4
Evaluating Cell Viability in μG Experiments
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To verify both the biocompatibility of MOC platform and assess viability of Human Glioblastoma A-172 and HUVEC cells after μG experiments, live imaging using LIVE/DEAD Viability/Cytotoxicity Kit (Sigma-Aldrich, Cat. No. L3224) was performed as per manufactures instructions. Briefly, fluorescent calcein-AM (2 μM) and red-fluorescent ethidium homodimer-1 (EthD-1) (4 μM) were gently injected into the MOC and incubated for 15 min at 37 °C and humidify atmosphere. MOCs were then washed with PBS and imaged using EVOS M5000 microscope (Invitrogen). To evaluate proliferation, cells were detached from the MOC using Trypsin-EDTA, collected, centrifugated and counted using a Hemocytometer. Trypan Blue 0.4% (Thermofisher Scientific, Cat. No. 15250061) was used to determine the percentage of viable cells present in the suspension.
5
Mitochondrial Activity and ROS Measurement
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RWPE-1 cells were plated on a chamber slide at a density of 1 × 105 cells/well the day before treatment. The RWPE-1 cells were treated with 10 nM DHT and/or 100 μM MitoQ for 24 h. Before incubating with fluorescent dye, RWPE-1 cells were treated with 0.6% H2O2 for 30 min. Mitochondrial activity and mtROS levels were measured by incubating cells with MitoTracker™ Green FM and MitoSOX™ Mitochondrial Superoxide Indicator (Invitrogen, MA, USA) for 30 min 37 °C. The slides were mounted and detected using an EVOS M5000 microscope (Invitrogen).
6
Calcium Imaging of Stretched 16HBE Cells
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16HBE cells were seeded at a density of 2 × 105 cells/well in complete growth medium in a 6-well plate for 24h. The following day, cells were treated with 100μM Dex for 24 h, and then cells in the stretch group were under mechanical stretch system for 1.5h. After cells were washed three times with PBS without Ca2+. Subsequently, all cells were incubated with 2.5μmol/L Fluo-4AM for 30 min at 37°C in the dark, then washed 3 times with PBS without Ca2+ to remove extracellular Fluo-4AM. Images were obtained using an EVOS M5000 microscope (Invitrogen by Thermo Fisher Scientific). The intensity of calcium fluorescence was quantified by Image-J software.
7
Visualizing Fungal Growth and Nuclei
8
Quantitative Dye Transfer Analysis
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For quantitative analysis of dye transfer and cell viability, images were acquired from 2 to 3 regions within each cut (6–9 measurements per well) using an EVOS M5000 microscope (Invitrogen, Waltham, MA, USA) equipped with a 10 × 0.3 NA air objective and EVOS LED light cubes for bright field imaging, FITC, RedX and DAPI. Images were acquired sequentially and exported to ImageJ for processing.
9
Quantification of DNA Damage in VSMC
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VSMC was cultured on glass coverslips until 70–75% confluent, followed by serum deprivation for a period of 48 h. VSMC was stimulated with EC EVs for a period of 24 hs followed by fixation with 3.7% paraformaldehyde solution and then washed 3x with PBS. Cells were made permeable through incubation with 0.1% Triton-PBS and blocked for 2 h at room temperature with 1% BSA-PBS-T (0.1% Tween). Cells were incubated with a rabbit antibody against Ser139 phosphorylated histone H2AX (γH2Ax) at 1 µg/mL overnight at 4°C. Cells were then washed with PBS 3x and incubated with goat anti-rabbit IgG Alexa Fluor 488 (Invitrogen A11034) at 1:1000 for 2 hours in the dark. After washing (3x with PBS), cells were mounted on glass slides with Prolong Gold w/Dapi reagent (ThermoFisher P36931). Cells were imaged at 40x magnification on EVOS M5000 microscope (Invitrogen). Accumulation of distinct foci within nucleus (>5) indicated a positive cell which were then counted as previously described [29 (link)].
10
Lung Tumor Infiltration Analysis
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As previously described in detail, lung samples were harvested 10 days after the second tumor challenge and fixed with 10% formalin. After rehydration with ethanol gradient solutions, the lungs were embedded in paraffin and sliced into 5-μm thick sections using a microtome. The sections were attached to a glass slide, rehydrated, and stained with hematoxylin and eosin. Any tumor infiltration in the lung was observed using an EVOS M5000 microscope (Invitrogen, Waltham, MA, USA).
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