50,000 cells were plated in a 12-well plate on a pre-autoclaved glass coverslip and incubated at 37C for 48 hours. Cell were then treated with a series of concentrations of SW044248 or DMSO for 4.5 hours, followed by treatment with 10 μM EdU or EU (Life Technologies Cat # C10337 or Cat # C10330)) and SW044248 for 1.5 hours. Cells were fixed with 3.7% formaldehyde in PBS for 20 min and Alexa dyes were conjugated to the alkyne-labeled EDu or EU by Click chemistry following the manufacturer’s instructions. Coverslips were mounted on slides in DAPI-containing mounting media (Sigma Cat # F6057). The cells were visualized and digital images collected with an EVOS-FL Cell Imaging System microscope (Life Technologies Cat # AMF4300) with a GFP and UV filter or Texas Red and UV filter.
Evos fl cell imaging system microscope
Manufactured by Thermo Fisher Scientific
Sourced in United States
The EVOS FL Cell Imaging System is a compact, automated microscope designed for live-cell imaging and analysis. It features LED-based illumination, high-resolution digital cameras, and user-friendly software for capturing and managing images of cells and other biological samples.
Automatically generated - may contain errors
Lab products found in correlation
C10337, by Thermo Fisher Scientific (1 mentions)
Amf4300, by Thermo Fisher Scientific (1 mentions)
F6057, by Merck Group (1 mentions)
Color ccd camera, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
4 protocols using evos fl cell imaging system microscope
1
Cell Proliferation Assay with EdU/EU
2
Immunofluorescence Staining of HFLS-RA Cells
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HFLS-RA cells were grown to confluency, and 10,000 cells were coated onto chamber slides. After 48 h at 37°C with 5% CO2, the medium was removed from each well and chilled, and enough methanol was added to cover the surface for fixing the cells. After incubating the slides for 15 min at -20°C, the methanol was removed, and PBS was used to hydrate the slides. The slides were then washed one more time with PBS. FITC-labeled CD58 mouse monoclonal antibody (Santa Cruz Biotechnology) was added to the cells and incubated for 1 h. After washing with PBS, cover slips were mounted, and the cells were visualized under a microscope. Images were taken at 40x magnification using the EVOS FL cell imaging system microscope with color CCD camera (Life Technologies, NY, USA).
3
Soft Agar Assay for Anchorage-Independent Growth
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The ability of FUT9 knockdown and control cells to grow in low‐anchorage conditions was determined by seeding cells in a soft agar medium. Cells were trypsinized and 2.5 × 104 cells suspended in 0.35% agar‐media supplemented with 10% (v/v) FBS and 4% (v/v) minimum essential medium vitamin solution (Life Technologies, 11120052) and layered on a 0.6% agar‐media bottom layer in 6 well plates. Cells were allowed to grow for 28 days and colonies were imaged using an EVOS FL Cell Imaging System microscope at 40× magnification (Life Technologies) and the density of colonies was quantified using ImageJ software.
4
Tumoursphere Propagation and Proliferation Assay
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Cells were seeded into 24- or 96-well Ultra-Low attachment plates (4 × 103 or 2 × 103 cells per well, respectively) in complete Mammocult medium (STEMCELL) and allowed to propagate in tumourspheres for 7 days. For each replicate, tumourspheres from 12 independent wells were combined and dissociated using Trypsin-EDTA (Gibco), and proliferation was assessed by cell counting. Images were obtained using EVOS FL Cell Imaging System microscope (Life Technologies).
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