We performed time-lapse acquisition with YouScope (http://langmo.github.io/youscope/ ) on Nikon-Ti Eclipse microscopes as described previously12 (link) with yellow fluorescent protein (500/20; 515LP; 535/30) and Cy5 (620/60; 660LP; 700/75; all, AHF) filter cubes to detect VENUS and LysobriteNIR, respectively. PCNAVENUS experiments were imaged every 20 minutes and trackSeq experiments every 30 minutes with 20× (NA 0.75) and 10× (NA 0.45) CFI Plan Apochromat λ objectives, respectively. Cells were cultured in Iscove modified Dulbecco medium12 (link) containing 16.6 μM LysobriteNIR (22641, AAT Bioquest), at 37°C, 5% oxygen, 5% carbon dioxide in SensoPlate 24 Well Glass bottom plates (Greiner Bio-One) for trackSeq experiments or μ-slide VI0,4 channel slides (IBIDI), coated with 2.5 or 10 μg/mL anti–CD43-biotin (eBIoR2/R60; eBioscience)14 (link) in phosphate-buffered saline at room temperature for 1 hour. The trackSeq experiments were analyzed by self-written automated live event recognition in time-lapse data (alerT) software, and others were tracked as described previously.6,15,17 (link)
Sensoplate 24 well glass bottom plates
Manufactured by Greiner
Sourced in Austria
SensoPlate 24 Well Glass bottom plates are designed for microscopy and imaging applications. These plates feature a glass bottom for improved optical clarity and performance. Each plate contains 24 individual wells.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using sensoplate 24 well glass bottom plates
1
Time-lapse imaging of VENUS and LysoBrite
2
CTC Enumeration Using IsoFlux System
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IsoFlux enriched CTCs were enumerated with an IsoFlux CTC enumeration kit containing all the staining antibodies (Fluxion Bioscience, Alameda, CA, USA). Briefly, after 10 min fixation with fixation buffer, cells were blocked for 10 min with 10% normal donkey serum (NDS) and probed with rabbit anti-human CD45 antibody (1:100) followed by detection with tetramethylrhodamine (TRITC) conjugated donkey-anti rabbit IgG (1:200). Cells were then washed twice with binding buffer and permeabilized using 0.2% Trition 100 before probing with fluorescein (FITC) conjugated anti-human Pan-cytokeratin antibody (1:10). After two further washes, cells were transferred to SensoPlate™ 24-well glass bottom plates (Greiner Bio-one, Kremsmünster, Austria) with Hoechst dye included in the mounting media. Fluorescent microscopy (ALS CellCelector™, ALS, Jena, Germany) was used for CTC enumeration.
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