Evos cell imaging system

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

The EVOS Cell Imaging System is a compact, automated microscope designed for live-cell imaging and analysis. It provides high-quality images and video capture of cells in a controlled environment. The system is equipped with advanced optics and illumination to enable clear visualization of cellular structures and processes.

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270 protocols using evos cell imaging system

Morphological Assessment of A375 Cells

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Morphological assessment of A375 cells was performed to detect the cellular changes induced by the tested compounds according to the method with slight modifications [37 (link)]. Briefly, 1×10⁴ cells were treated with or without the compounds in a sterile chamber slide system (Nunc™ Lab-Tek™ II Chamber Slide™ System) for 48h. Prior cellular examination, the media was removed and replaced with PBS. Treated cells with typical morphological changes of apoptosis were imaged using phase contrast inverted microscope (EVOS cell imaging system, Thermo Fisher Scientific, MA, USA).
A375 cells were examined with fluorescence microscope using ReadyProbes^® cell viability imaging kit according to manufacturer's recommendations (Thermo Fisher Scientific, MA, USA). A375 cells were treated in the same condition as mentioned in the previous section. Both dyes were added for 15 mins and slides were visualised using EVOS cell imaging system (Thermo Fisher Scientific).

Al-Qathama A., Gibbons S, & Prieto J.M. (2017). Differential modulation of Bax/Bcl-2 ratio and onset of caspase-3/7 activation induced by derivatives of Justicidin B in human melanoma cells A375. Oncotarget, 8(56), 95999-96012.

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Autophagy Quantification in C. elegans

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To measure the number of autophagosomes in vivo we used the DA2123 (adIs2122 [lgg-1p::GFP::lgg-1 + rol-6(su1006)]) strain. We quantified the GFP positive puncta in the seam cells of adult day 1 animals using the 60x lens of a widefield epifluorescence microscope (EVOS Cell Imaging Systems, Thermo Fisher Scientific, Waltham, MA, USA). Additionally, protein from whole animal extracts was used to quantify the levels of unlipidated and lipidated LGG-1 (the C. elegans ortholog of LC3/Atg8) under the tested conditions. Additionally, we calculated SQST-1::GFP (the C. elegans ortholog of SQSTM1) positive puncta using the HZ589: bpIs151[sqst-1p::sqst-1::GFP + unc-76(+)] strain. Puncta were measured in the head region or in the seam cells of the animals. Fluorescent images were acquired using the EVOS Cell Imaging Systems (Thermo Fisher Scientific, Waltham, MA, USA). For the assessment of autophagy flux, we used the DLM1 reporter strain bearing the transgene (eft-3p::CERULEAN-VENUS::lgg-1 + unc-119(+)). Processing of dual fluorescent protein fused to LGG-1 (dFP::LGG-1) upon fusion with the lysosome led to the production of monomeric fluorescent protein (mFP), detected by anti-GFP western blot.

Gkikas I., Daskalaki I., Kounakis K., Tavernarakis N, & Lionaki E. (2023). MitoSNARE Assembly and Disassembly Factors Regulate Basal Autophagy and Aging in C. elegans. International Journal of Molecular Sciences, 24(4), 4230.

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Cre-dependent eGFP Expression System

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Stable expression system of Cre recombinase-dependent eGFP was established by transfecting HEK293T cells with pLV-CMV-LoxP-DsRed-LoxP-eGFP^{57 (link)} (a gift from J. Rheenen), with puromycin selection following a protocol from the Genetic Perturbation Platform at Broad Institute. The selected cells were confirmed with lack of eGFP by EVOS Cell Imaging Systems (Thermo Fisher Scientific). Cells were seeded in 96-well plates at 1.6 × 10⁴ cells per well and after 24 h transfected with in vitro-transcribed cre mRNA (12.5 ng) and in vitro-transcribed tS variants (25 ng) pairing to different PTCs (tS::UGA, tS::UAG; tS::UAA) using MessengerMax (0.2 µl per well). Every day within four days after transfection the fluorescence was recorded on Spark microplate reader (Tecan) and reported as relative fluorescence units. The expression of DsRed and eGFP were visualized by EVOS Cell Imaging Systems (Thermo Fisher Scientific).

Albers S., Allen E.C., Bharti N., Davyt M., Joshi D., Perez-Garcia C.G., Santos L., Mukthavaram R., Delgado-Toscano M.A., Molina B., Kuakini K., Alayyoubi M., Park K.J., Acharya G., Gonzalez J.A., Sagi A., Birket S.E., Tearney G.J., Rowe S.M., Manfredi C., Hong J.S., Tachikawa K., Karmali P., Matsuda D., Sorscher E.J., Chivukula P, & Ignatova Z. (2023). Engineered tRNAs suppress nonsense mutations in cells and in vivo. Nature, 618(7966), 842-848.

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Evaluating HaCaT Cell Viability with H2O2 Exposure

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The viability of H₂O₂-treated HaCaT cells was measured by a CTG 2.0 assay using a previously reported method (Ma et al., 2016 (link); Ma et al., 2018 (link)). HaCaT cells were seeded in 96-well plates at 5×10³ cells per well and allowed to attach for 12 h. Media was then removed and 100 μL of H₂O₂ (at concentrations of 50, 100, 200, 400, and 800 μM) was added and incubated for 24 h. Next, CTG 2.0 reagent (100 μL) was added in each well and shaken at 200 rpm for 2 min on an orbital shaker. The plate was then kept at room temperature for 10 min after which luminescence intensity was recorded using a Spectramax M2 plate reader. Morphological analysis was conducted to evaluate cell damage with crystal violet staining method. Cells were fixed in 70% ethanol for 15 min after which medium was removed. Staining solution (0.05% w/v) was added to each well and incubated for 20 min. Then the staining solution was removed and cells were washed with PBS for three times. The morphological changes of cells were observed by an EVOS Cell Imaging System (Invitrogen, Waltham, MA, USA).

Liu C., Guo H., DaSilva N.A., Li D., Zhang K., Wan Y., Gao X.H., Chen H.D., Seeram N.P, & Ma H. (2019). Pomegranate (Punica granatum) Phenolics Ameliorate Hydrogen Peroxide-Induced Oxidative Stress and Cytotoxicity in Human Keratinocytes. Journal of functional foods, 54, 559-567.

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3D Fibrin Gel Assay for Angiogenic Potential

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3D fibrin gel assay was performed as previously described^{25 (link)}. Briefly, Cytodex® 3 microcarrier beads (Sigma) were incubated with HUVEC, HUVEC-TIE2-WT or HUVEC-TIE2-L914F at a concentration of 400 cells/ bead for 4 h at 37 °C. The following day, coated beads were resuspended in 2 mg/mL of fibrinogen (Sigma) solution containing 0.15 U/mL of aprotinin (Sigma) at a concentration of 500 beads/ml. Then 0.625 U/mL of thrombin (Sigma) and 0.5 ml beads/fibrinogen suspension were added per well of a 24-well plate and incubated at 37 °C to allow fibrin clotting. The gels were overlaid with human lung fibroblasts at 2 × 10⁴ cells/well and medium was replaced every other day. Where indicated fibroblasts were omitted and EGM2/10% FBS medium substituted with EBM2 (Endothelial basal medium) which does not contain growth factors. In assays conduced with pericytes the Cytodex® beads were seeded with EC (HUVEC or HUVEC-TIE2-L914F) and pericytes at a 10:1 ratio. Images were acquired with the Nikon A1R LUN-V Inverted Microscope and EVOS cell imaging system (Invitrogen) and analyzed with Nikon NIS-Elements and ImageJ.

Cai Y., Schrenk S., Goines J., Davis G.E, & Boscolo E. (2019). Constitutive Active Mutant TIE2 Induces Enlarged Vascular Lumen Formation with Loss of Apico-basal Polarity and Pericyte Recruitment. Scientific Reports, 9, 12352.

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Immunofluorescent Staining of Cultured Fibroblasts

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Cultured and treated primary capsular fibroblasts were fixed using 100% methanol fix for 15 minutes. After fixing, cells were rinsed in 1× PBS. Cells were then blocked using 5% BSA in 1× PBS for 1 hour with gentle rocking at room temperature. Cells were incubated with primary αSMA antibody (rabbit, 1:1,000, Abcam, Cambridge, UK) for 1 hour at room temperature (RT). After primary incubation, antibody solution was removed and washed with 1× PBS 3 times. Cells were then incubated with secondary antibody (AlexaFluor 647 conjugated goat antirabbit, 1:500, ThermoScientific, Waltham, MA) for 45 minutes at RT. The secondary solution was removed and then washed 3 times with 1× PBS before visualizing with an EVOS cell imaging system (Invitrogen, Carlsbad, CA).

Park R.H., Pollock S.J., Phipps R.P., Langstein H.N, & Woeller C.F. (2019). Discovery of Novel Small Molecules that Block Myofibroblast Formation: Implications for Capsular Contracture Treatment. Plastic and Reconstructive Surgery Global Open, 7(9), e2430.

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Apoptosis Detection in Poppy Leaves

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The infected and healthy poppy leaf samples were cut into small pieces, washed twice with PBS, and immediately fixed in paraformaldehyde (4%; w/v), and then kept in 100 mM phosphate buffer (pH 7.2) overnight. After dehydration of the samples in graded series of ethanol, they were embedded in paraffin. The sections were cut at 8 μm thickness and mounted on slides. Sections were rehydrated and incubated with proteinase-K for 15 min at 37°C. The TUNEL reaction solution (Invitrogen, United States) was mixed with the dried sample. After incubation at 37°C for 1 h, sections were rinsed three times with PBS and examined at 560 nm as a greenish fluorescence, and photographed at 40× magnification with an EVOS Cell Imaging System (Invitrogen, United States).

Srivastava A., Agrawal L., Raj R., Jaidi M., Raj S.K., Gupta S., Dixit R., Singh P.C., Tripathi T., Sidhu O.P., Singh B.N., Shukla S., Chauhan P.S, & Kumar S. (2017). Ageratum enation virus Infection Induces Programmed Cell Death and Alters Metabolite Biosynthesis in Papaver somniferum. Frontiers in Plant Science, 8, 1172.

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Oxidative Stress-Induced Apoptosis Assay

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HaCaT cells were seeded in 24 well plates at 0.5×10⁵ cells per well and allowed to attach for 12 h followed by treatment of test samples (at aforementioned concentrations) for 6 h. Medium were then removed and cells were washed twice with PBS. Next, 300 μL of fresh medium containing H₂O₂ (200 μM) were added to cells and incubated for 24 h. Medium were then removed and cells were washed twice with PBS. Hoechst 33342 staining buffer (300 μL) was added to the cells and incubated for 30 min in darkness. Next, PI staining buffer (300 μL) was added to the cells and incubated for 30 min in darkness. The morphological changes of the nucleus of the cells were observed by an EVOS Cell Imaging System with a fluorescence microscope (Invitrogen, Waltham, MA, USA).

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Quantifying Intracellular and Mitochondrial ROS

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The production of intracellular and mitochondrial ROS was evaluated using DCF-DA (Sigma-Aldrich Chemical Co.) and MitoSOX™ red dye (Thermo Fisher Scientific), respectively. The cells were treated with coptisine for 30 min, and then incubated with 10 μM DCF-DA or 1 μM MitoSOX™ red, for 20 min, at 37 °C, without light. The fluorescence intensity was detected by flow cytometry. For image analysis of the ROS production, the cells were collected using a Thermo Shandon Cytospin 3 cytocentrifuge (Marshall Scientific, Hampton, NH, USA), and stained with DAPI, which is a nuclear-specific fluorescence dye. Subsequently, the cells were mounted using a mounting solution (Sigma-Aldrich Chemical Co.), and the images were visualized using an EVOS Cell Imaging System (Invitrogen, Carlsbad, CA, USA).

Kim S.Y., Hwangbo H., Lee H., Park C., Kim G.Y., Moon S.K., Yun S.J., Kim W.J., Cheong J, & Choi Y.H. (2020). Induction of Apoptosis by Coptisine in Hep3B Hepatocellular Carcinoma Cells through Activation of the ROS-Mediated JNK Signaling Pathway. International Journal of Molecular Sciences, 21(15), 5502.

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Fluorescent Staining and Imaging of Cells

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Homogeneous samples containing medium, microcarriers and cells were stained with Hoechst (dilution 1:626, 33342 392/440, ThermoFisher) and Ethidium homodimer (dilution 1:300, E1169, Invitrogen™) and imaging was performed with the use of an EVOS™ Cell Imaging System (M5000, Invitrogen™).

Hubalek S., Melke J., Pawlica P., Post M.J, & Moutsatsou P. (2023). Non-ammoniagenic proliferation and differentiation media for cultivated adipose tissue. Frontiers in Bioengineering and Biotechnology, 11, 1202165.

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