Imagexpress micro xl high content screening system

Manufactured by Molecular Devices

Sourced in United States

The ImageXpress Micro XL High-Content Screening System is a fully automated, high-throughput microscope designed for cell-based assays and high-content analysis. It features a motorized stage, autofocus capabilities, and multiple imaging channels to capture images of cells and other samples.

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Lab products found in correlation

Hoechst 33342, by Thermo Fisher Scientific (10 mentions) 5 fluoro 2 deoxyuridine fudr, by Merck Group (2 mentions) Tubercidin, by Merck Group (1 mentions) Culture media, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Glass bottom 96 well plate, by Greiner (1 mentions) Metaxpress high content image acquisition analysis software, by Molecular Devices (1 mentions) Calcein acetoxymethyl ester am, by Merck Group (1 mentions) Celltiter 96 aqueous one solution cell proliferation kit, by Promega (1 mentions) Click ittm edu imaging kit, by Thermo Fisher Scientific (1 mentions) Metaxpress software, by Molecular Devices (1 mentions)

10 protocols using imagexpress micro xl high content screening system

High-Throughput Cytotoxicity Screening Assay

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The
cells were seeded in 384-well plates (Corning, Cat. No. 3712),
containing 45 μL of the cell suspension in culture media (Gibco)
with 10% fetal bovine serum (FBS, Gibco) at a density of 800 cells
per well. Cells were incubated in a humidified atmosphere with 5%
CO₂. Stock solutions of test compounds were prepared in
DMSO (Sigma-Aldrich, Germany). The next day (overnight incubation),
cells were treated for 72 h with test compounds titrated in DMSO (the
final concentration of DMSO did not exceed 0.5%). Tubercidin (Sigma-Aldrich)
served as a positive control. After 72 h, 5-ethynyl-2′-deoxyuridine
(EdU) diluted in PBS was added to cells and incubated for 2.5 h in
a 5% CO₂ humidified atmosphere at 37 °C. Cells were
then fixed in 4% paraformaldehyde for 30 min at room temperature and
stained with Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA).
After rinsing four times with PBS, the Click-iT reaction with Alexa
Fluor 488 dye was performed for 1 h at room temperature (protected
from light), followed by rinsing four times with PBS for further imaging.
Imaging and analysis were performed using an ImageXpress Micro XL
High-Content Screening System (Molecular Devices LLC, San Jose, CA).
IC₅₀ values were determined by sigmoidal curve fitting
using GraphPad Prism 6 software.

Tsyganov D.V., Samet A.V., Silyanova E.A., Ushkarov V.I., Varakutin A.E., Chernysheva N.B., Chuprov-Netochin R.N., Khomutov A.A., Volkova A.S., Leonov S.V., Semenova M.N, & Semenov V.V. (2022). Synthesis and Antiproliferative Activity of Triphenylphosphonium Derivatives of Natural Allylpolyalkoxybenzenes. ACS Omega, 7(4), 3369-3383.

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HSP-4 reporter assay for ER stress

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The assay was carried out as previously described^{56 (link)}. In brief, age-synchronized SJ4005 hsp-4::gfp reporter animals were cultured at 20 °C in 96-well plates containing S. complete media and irradiated Op50 bacteria (6 mg/mL). To prevent self-fertilization, 5-fluoro-2’- deoxyuridine (FUDR, 0.12 mM final) (Sigma, cat # 856657) was added at the L4 developmental stage. 24 hours before imaging, JZL184 (between 2 – 50 μM) was added to the worms. On day 1, worms in the positive control group were treated with 5 μg/ml tunicamycin and all worms were imaged 8 hours later. GFP fluorescence was imaged with ImageXpress Micro XL High-Content screening system (Molecular Devices) and a 2× objective.

Chen A.L., Lum K.M., Gonzalez P.L., Ogasawara D., Cognetta AB I.I.I., To A., Parsons W.H., Simon G.M., Desai A., Petrascheck M., Bar-Peled L, & Cravatt B.F. (2019). Pharmacological convergence reveals a lipid pathway that regulates C. elegans lifespan. Nature chemical biology, 15(5), 453-462.

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HSP-4 reporter assay for ER stress

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Quantifying Wound Healing Potential

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Parental A549 and H1299 cells and their IR-surviving sublines were seeded in a 96-well plate at the density of 2 × 10⁴ cells per well and incubated at 37 °C, 5 % CO₂ for 72 h to create a confluent monolayer. The cell monolayer was then scrapped in a straight line to create a “scratch” with a sterile micropipette tip and washed with PBS (pH = 7.4) 3 times to remove cell debris. Cells were incubated with the complete culture medium for another 72 h. Images of initial areas of the wounds were captured at time zero (t = 0 h) and at 24 h, 48 h, and 72 h (t = Δ h) after scratching to observe the cell migration toward the wounded area. The images were captured using an ImageXpress Micro XL High-Content Screening System (Molecular Devices LLC, San Jose, CA, USA). The wound areas were calculated using the Custom Module Editor of the MetaXpress 5.0 Software. The migration ability of cells was expressed as a percentage of wound closure using the following equation:
where A(0) is the initial area of wound immediately after scratching, and A(t) is the wound area measured at 24 h, 48 h or 72 h after scratching.

Alhaddad L., Pustovalova M., Blokhina T., Chuprov-Netochin R., Osipov A.N, & Leonov S. (2021). IR-Surviving NSCLC Cells Exhibit Different Patterns of Molecular and Cellular Reactions Relating to the Multifraction Irradiation Regimen and p53-Family Proteins Expression. Cancers, 13(11), 2669.

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Quantifying Cell Proliferation with EdU

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Cells (at concentrations of 15 × 10² cells/0.32 cm² and 2 × 10³ cells/0.32 cm²) were seeded in a 96-well plate for 72 h. Then, 5-ethynyl-2-deoxyuridine (EdU) labeling reagent (final concentration 10 μM) was added to cell cultures and maintained in a 5% CO₂ humidified incubator at 37 °C for 2.5 h. Then, cells were fixed in 2% (v/v) paraformaldehyde at room temperature for 15 min and incubated with 6 μg/mL Hoechst 33,342 (Thermo Fisher Scientific, Waltham, MA, USA) overnight for nuclear staining. Following two rinses in 1XPBS, 1XEdU buffer additive was added for 1 h and incubated at room temperature protected from light. Imaging and analysis of proliferating cells were performed using the ImageXpress Micro XL High-Content Screening System (Molecular Devices LLC, San Jose, CA, USA).

Alhaddad L., Chuprov-Netochin R., Pustovalova M., Osipov A.N, & Leonov S. (2023). Polyploid/Multinucleated Giant and Slow-Cycling Cancer Cell Enrichment in Response to X-ray Irradiation of Human Glioblastoma Multiforme Cells Differing in Radioresistance and TP53/PTEN Status. International Journal of Molecular Sciences, 24(2), 1228.

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High-Content Screening of Radiation-Induced Proliferation

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Cells (at concentrations of 2000/0.056 cm²) were seeded in a 384-well plate for 24 h and 48 h following exposure to an extra single dose of 5 Gy. Then, EdU labeling reagent (final concentration 10 μM) was added to cell cultures and maintained in a 5% CO₂ humidified incubator at 37 °C for 2.5 h. Then, cells were fixed in 2% (v/v) paraformaldehyde at room temperature for 15 min and incubated with 6 μg/mL Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA) overnight for nuclei staining at 4 °C. Following two rinses in 1× PBS, 1× EdU buffer additive was added for 1 h and incubated at room temperature protected from light. Imaging and analysis of proliferating cells were performed using the ImageXpress Micro XL High-Con-tent Screening System (Molecular Devices LLC., San Jose, CA, USA).

Pustovalova M., Blokhina T., Alhaddad L., Chigasova A., Chuprov-Netochin R., Veviorskiy A., Filkov G., Osipov A.N, & Leonov S. (2022). CD44+ and CD133+ Non-Small Cell Lung Cancer Cells Exhibit DNA Damage Response Pathways and Dormant Polyploid Giant Cancer Cell Enrichment Relating to Their p53 Status. International Journal of Molecular Sciences, 23(9), 4922.

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Jurkat T Cell Binding Assay

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Glass-bottom 96-well plates (Greiner Bio-One, Morone, NC) were filled with 1 M NaOH for 1 h, washed several times with ultra-pure distilled water and then dried by a stream of nitrogen gas. Planar lipid bilayers decorated with scFv ligands were assembled in wells. Jurkat T cells were stained with 5 µM calcein-acetoxymethyl ester (AM) (Sigma-Aldrich, St. Louis, MO, USA) for 45 min at 37°C. 1 × 10⁵ cells were added per well and incubated at 37°C for 30 min. Unbound cells were removed by serial washes with PBS. The number of bound cells was measured at four sites per well using an ImageXpress Micro XL High-Content Screening System (Molecular Devices, CA, USA). Counts from eight sites (two wells) per each group were analyzed using MetaXpress High-Content Image Acquisition & Analysis Software (Molecular Devices, CA, USA).

Al-Aghbar M.A., Chu Y.S., Chen B.M, & Roffler S.R. (2018). High-Affinity Ligands Can Trigger T Cell Receptor Signaling Without CD45 Segregation. Frontiers in Immunology, 9, 713.

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Cell Viability and Cell Cycle Assay

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Cell viability was assessed using the CellTiter 96^® AQueous One Solution Cell Proliferation kit (Promega) on a Multiscan Spectrum spectrophotometer (Thermo LifeSciences), with absorbance read at 490 nm.
For cell cycle progression using EdU, MCF10A cells were seeded in 384-well plates at a density of 800 cells per well. After spreading, cells were treated for 18 h with compounds in DMSO (final DMSO concentration did not exceed 0.5%). EdU from kit C10337 (ThermoFischer Scientific, Waltham, MA, United States) was then added to the cells at 10 μM and incubated for 2.5 h. Cells were fixed in 4% paraformaldehyde for 30 min at room temperature. Click-iT reaction was performed for 1 h at room temperature, protected from light, with 5 µM Alexa Fluor 488 azide in (100 mM Tris, 1 mM CuSO4, 50 mM Ascorbic Acid, pH 8.5). Nuclei were counterstained with Hoechst 33,342 (Thermo Fisher Scientific). Imaging and analysis were performed using the ImageXpress Micro XL High-Content Screening System (Molecular Devices LLC, San Jose, CA, United States). IC50 values were determined by sigmoidal curve fitting using GraphPad Prism six software.

Fokin A.I., Chuprov-Netochin R.N., Malyshev A.S., Romero S., Semenova M.N., Konyushkin L.D., Leonov S.V., Semenov V.V, & Gautreau A.M. (2022). Synthesis, Screening and Characterization of Novel Potent Arp2/3 Inhibitory Compounds Analogous to CK-666. Frontiers in Pharmacology, 13, 896994.

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Quantifying S-Phase Cell Proliferation

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S phase cell proliferation analysis was performed using the Click-iTTM EdU Imaging Kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Cells (at concentrations of 1.5 × 10³ and 2 × 10³ cells/0.32 cm²) were seeded into a 96-well plate. After three days, 2 × EdU solution was added to cells and incubated for 2.5 h at 37 °C and 5% CO₂ in humidified conditions. Cells were then fixed in 2% paraformaldehyde (PFA) for 15 min at room temperature and incubated with 40 µM Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA) for nuclei staining, protected from light. Following two rinses with PBS, 1X Click-iT^® EdU buffer additive was added for one hour and incubated at room temperature protected from light. Imaging and analysis of proliferating cells were performed using the ImageXpress Micro XL High-Content Screening System (Molecular Devices LLC, San Jose, CA, USA).

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Quantification of Phospho-CDC6 Foci

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After AZD5153 treatment, U2OS cells were pre-extracted with 0.5% triton in PBS for 5 min before being fixed with 4% paraformaldehyde for 15 min at room temperature. Cells were then blocked with 5% BSA in PBST followed by primary antibody (Phospho-CDC6 S54, Abcam # ab75809, 1:500) and secondary antibody (Alexa Fluor 488 conjugate) staining. DAPI was used to counter stain DNA. Image acquisition was carried out on ImageXpress MicroXL High Content Screening System (Molecular Devices, Sunnyvale, CA, USA). The number of phospho-CDC6 foci in the nuclei was scored and quantified using MetaXpress Software (Molecular Devices). An average of 500 nuclei were analyzed for each experimental condition. Results shown represent average of three independent experiments.

Zhang J., Dulak A.M., Hattersley M.M., Willis B.S., Nikkilä J., Wang A., Lau A., Reimer C., Zinda M., Fawell S.E., Mills G.B, & Chen H. (2018). BRD4 facilitates replication stress-induced DNA damage response. Oncogene, 37(28), 3763-3777.

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