The cPass neutralization assay was performed with mouse sera, according to the manufacturer’s instructions (Genscript). A SARS-CoV-2 pseudo-virus neutralization assay was performed with rat sera at the Israeli Central Virology Lab (Sheba Medical Center, Tel Hashomer, Israel). The propagation-competent vesicular stomatitis virus expressing cSARS-CoV-2 S protein and carrying the gene encoding green fluorescence protein (psSARS-2) was used in an assay similar to a recently reported assay [33] (link) shown to correlate well with an authentic SARS-CoV-2 virus micro-neutralization assay. Following titration, 100 focus forming units (ffu) of psSARS-2 were incubated with 2-fold serial dilutions of heat-inactivated (56 °C, 30 min) immune rat sera. After incubation for 60 min at 37 °C, the virus/serum mixtures were transferred to Vero E6 cells that had been grown to confluence in 96-well plates, and incubated for 90 min at 37 °C. Thereafter, 1% methyl cellulose in Dulbecco's modified eagle's medium (DMEM) with 2% fetal bovine serum (FBS) was added, and plates were incubated for 24 h, after which, 50% plaque reduction titre was calculated by counting green fluorescent foci using a fluorescence microscope (EVOS M5000, Invitrogen).
Evos m5000
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
The EVOS M5000 is a compact and user-friendly inverted fluorescence microscope designed for live-cell imaging. It features a high-resolution CMOS camera, LED illumination, and an intuitive touch-screen interface for easy operation. The EVOS M5000 is suitable for a variety of applications, including cell culture monitoring, time-lapse imaging, and fluorescence-based assays.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (5 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (4 mentions)
Shandon cytospin 4, by Thermo Fisher Scientific (3 mentions)
Tumor necrosis factor (tnf), by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 conjugated antibody, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Fluorescein isothiocyanate (fitc), by Merck Group (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Mitosox, by Thermo Fisher Scientific (2 mentions)
Plasmocin, by InvivoGen (2 mentions)
Jc 1 mmp assay kit, by Beyotime (2 mentions)
Mitosox red mitochondrial superoxide indicator, by Thermo Fisher Scientific (2 mentions)
Facscanto 2, by BD (2 mentions)
Evos m5000 microscope, by Thermo Fisher Scientific (2 mentions)
Image pro plus program, by Media Cybernetics (2 mentions)
Cm1860, by Leica (2 mentions)
Masson staining, by Solarbio (2 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (2 mentions)
S302380, by Agilent Technologies (2 mentions)
Matrigel matrix, by Corning (2 mentions)
196 protocols using evos m5000
1
SARS-CoV-2 Pseudovirus Neutralization Assay
2
Quantifying Hepatocyte Proliferation
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Two hours prior to euthanasia, mice were administered 5′-bromo-2′-deoxyuridine (100 mg/kg; Sigma) i.p. Formalin fixed paraffin embedded (FFPE) liver sections were incubated with primary antibody at 4 °C overnight. Secondary antibody was applied for 1.5 h at room temperature, and nuclei were counterstained with DAPI. Ten high-powered fields (400×) were visualized, and 5-bromo-2′-deoxyuridine (BrdU)-positive hepatocytes were counted on an immunofluorescence microscope (Invitrogen EVOS M5000).
3
Culturing HEK293, HEK293-derived, and HaCaT Cells
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HEK293, HEK293-derived stable cells, and HaCaT cells were cultured in DMEM supplemented with 10% fetal bovine serum (Welgene) and 1× penicillin-streptomycin (Welgene). All cells were grown at 37°C with 5% CO2 under humidified conditions. For transfection, the Gene-Fect Transfection Reagent (Translab) was used following the manufacturer’s instructions. CELENA S (Logos Biosystems) and EVOS M5000 (Invitrogen) were used for the fluorescence imaging of living cells.
4
Breast Cancer Spheroid Assay
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Cell lines were automatically counted with the BIORAD TC20 automated cell counter (Bio-Rad, Hercules, CA, USA) and 10,000 cells were plated in 200 µL of fresh media containing 10 nM E2 or EtOH in Ultra-Low Attachment (ULA) 96-well plates (Corning, Somerville, MA, USA). Pictures were taken with the EVOS™ M5000 imaging system (Invitrogen™, Thermo Fisher Scientific, Waltham, MA, USA) every 24 h for 5 days. The spheroid size was measured with ImageJ, and the average size relative to the respective negative control at day 1 was calculated. The experiments were performed in triplicate and repeated three times independently.
5
Perfusion-based Tissue Fixation and Cryosectioning
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Four months postinjection, mice were euthanized by transcardial perfusion under terminal anesthesia. In short, anesthetized mice were fixed in a supine position, and a thoracotomy was performed. First, 10 ml of PBS followed by 10 ml of 4% PFA was perfused into the left ventricle, and the right atrium was resected. As the fixative (PFA) enters, the body stiffens, and the liver turns pale, indicating a successful perfusion procedure. After perfusion, organs were harvested and incubated in 30% sucrose overnight at 4°C. The next day, organs were embedded in the optimal cutting temperature medium and stored at -80°C until cryo-sectioned. Cryo-sections (12 mm) were made with a cryostat microtome system (Leica, Germany) and stored at -20°C before antibody staining. Sections were permeabilized with 1x PBS containing 0.2% Triton-X for 20 minutes, stained with nuclear stain DAPI (1 in 10,000), and washed with 1xPBS (3 times) for 15 mins. Slides were imaged for native GFP signal and DAPI nuclear stain on a fluorescence microscope (EVOS M5000, Invitrogen).
6
Immunofluorescence Staining of YB-1
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1 × 104 cells were grown directly on coverslips and treated with the inhibitors. 24 h after treatment, cells were fixed using ice-cold methanol-acetone (1:1) for 15 min at −20 °C. Fixed cells were blocked with 3% BSA (in PBS) and stained for YB-1 (Abcam, #EP2706Y) followed by incubation with secondary anti-rabbit Alexa Fluor 488-conjugated antibody (Invitrogen, #A11008). Slides were mounted with ProLong® Gold Antifade Mountant with DAPI (Thermo Fisher, #P36931). Images were taken with the AxioVert.A1 Microscope and Camera AxioCam ERc5s (Zeiss) or an Evos M5000 from Invitrogen.
7
Osteogenic Differentiation of Mesenchymal Stem Cells
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MSCs were differentiated into osteogenic lineages, for which cells (P3) were seeded in triplicate, one as a negative control, onto 12-well plates at a density of 2 × 104 cells/well. After 48 h, the medium was switched to an inducing medium (StemPro Osteogenesis Differentiation Kit) or maintained in regular growth medium for a negative control sample. After 21 days, cells were fixed in 4% paraformaldehyde and Ca2+ deposits stained with 1% Alizarin Red S (Acros Organics, New Jersey, United States). The plates were analyzed using an inverted microscope (EVOS M5000, Invitrogen).
8
Visualizing Mitochondria in Fusarium
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F. oxysporum cells expressing FoSir5-GFP or FoDLAT-GFP fusion proteins were incubated on PDA plates at 25°C for 3 days. The mycelia of the tested strains were then collected and preincubated for 15 min with 200 nM MitoTracker Red CMRos (M7512, Invitrogen). After washing with phosphate-buffered saline (PBS), pH 7.4, the mycelia were stained with 1 μg/ml DAPI (D9542, Sigma) at room temperature in darkness for 5 min, followed by washing with PBS three times. Fluorescence microscope was performed using microscope of EVOS M5000 (Invitrogen).
9
Wound Closure Assay with E2 Treatment
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Cell lines were automatically counted with the BIORAD TC20 automated cell counter (Bio-Rad, Hercules, CA, USA) and 25,000 cells were plated in 100 µL of fresh media in 2 chamber wells (Ibidi GMBH, Martinsried, Planegg, Germany). Cells were then incubated at 37 °C with 5% CO2. After 24 h, the chambers were removed, and the culture media were replaced with fresh media containing 10 µM mitomycin C to stop cell proliferation and 10 nM E2 or EtOH. Pictures were taken with the EVOS™ M5000 imaging system (Invitrogen™, Thermo Fisher Scientific, Waltham, MA, USA) immediately (0 h) and every 6 to 12 h until the gap between the cells was filled. The wound area was measured with ImageJ and the percentage of wound area was calculated relative to the wound area at 0 h of migration. The experiments were performed in triplicate and repeated three times independently.
10
Transduction of 293T/17 and HeLa Cells
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293T/17 cells or HeLa cells (ATCC #CCL-2) were seeded in 24-well plates, and, when they had reached a confluency of approximately 60%–70%, cells were transduced with rAAV at the MOI as indicated. Images were taken 3 days post transduction using a microscope with a built-in camera (Evos M5000, Invitrogen).
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