pEGFP was a kind gift from Assoc. Prof. Masayuki Takahashi (Hokkaido University). Cells were transfected with plasmid DNA using Superfect Transfection Reagent (Qiagen, Germany) in DMEM according to the manufacturer’s protocol. Transfected cells were replated onto a glass-bottom 96-well plate (Corning, USA) precoated with 0.1 mg/mL poly-D-lysine for live cell imaging.
Glass bottom 96 well plate
Manufactured by Corning
Sourced in United States
Glass-bottom 96-well plates are a type of laboratory equipment used for various cell culture and imaging applications. These plates feature a transparent glass bottom that allows for direct observation and analysis of cells or samples under a microscope. The 96-well format provides a standardized platform for high-throughput experiments and assays.
Automatically generated - may contain errors
Lab products found in correlation
Cellasic system, by Merck Group (2 mentions)
Superfect transfection reagent, by Qiagen (1 mentions)
Deltavision elite wide field fluorescence microscope, by GE Healthcare (1 mentions)
Softworx software, by GE Healthcare (1 mentions)
Inverted light microscope, by Nikon (1 mentions)
Dmil microscope, by Leica (1 mentions)
Victor3 multilabel plate reader, by PerkinElmer (1 mentions)
Passive lysis buffer (plb), by Promega (1 mentions)
Luciferin, by Promega (1 mentions)
6 protocols using glass bottom 96 well plate
1
Transient Expression of pEGFP
2
Live Imaging of Engineered Neural Assembloids
3
Live-cell imaging of meiotic cells
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Images were acquired using a DeltaVision Elite wide-field fluorescence microscope (GE Healthcare) and a PCO Edge scientific complementary metal–oxide–semiconductor camera, with softWoRx software and a 60× NA1.42 oil-immersion Plan Apochromat objective. GFP and RFP filter sets were used, with the setting information for acquiring each figure panel provided in Table S4 . Live-cell imaging was performed exactly as described in King et al. (2019) (link), except fresh SPO was used in place of conditioned SPO. In short, cells were imaged in an environmental chamber heated to 30°C, using either the CellASIC ONIX Microfluidic Platform or concanavalin A–coated glass-bottom 96-well plates. Cultures of meiotic cells in SPO were transferred to the microfluidic plates and loaded at 8 psi for 5 s. SPO was applied with a constant flow rate pressure of 2 psi for 15–20 h. With plates, cells were adhered to wells, and 100 µl of SPO was added to each well. Specific imaging conditions are noted in Table S4 . All time-lapse experiments were performed using the CellASIC system (EMD Millipore) in Y04D or Y04E microfluidics plates, with the exception of the LatA experiments, for which we used glass-bottom 96-well plates (Corning). Images were deconvolved using softWoRx software (GE Healthcare) using 3D iterative constrained deconvolution with 15 iterations and enhanced ratio.
4
Spheroid Outgrowth and Adhesion Analysis
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The aggregation of cells to spheroids was observed qualitatively at 20× magnification on a Nikon inverted light microscope in ultra-low adherence plates. Spheroid outgrowth was determined after plating on glass-bottom 96-well plates (Corning) for 4, 8, and 12 h by imaging on an inverted Leica DmiL microscope at 10× magnification. Adhesion capacity was depicted through promotion of outgrowth protruding from the primary spheroids.
5
AAV Neutralizing Antibody Screening Protocol
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Prior to vector delivery, all animals in the AAV study arm were screened for neutralizing antibodies using previously described methods (16 (link)). Specifically, 25 μL of porcine serum was mixed with an equal volume containing recombinant AAV9 (1,000–10,000 vg/cell) packaging ssCBA-Luc transgenes prediluted in DMEM + 5% FBS + penicillin-streptomycin. The mixture was added to tissue culture-treated, black, glass- bottom 96-well plates (Corning) and incubated at room temperature for 30 min. A total of 5 × 104 HEK293 cells in 50 μL of medium was then added to each well, and the plates were incubated in 5% CO2 at 37°C for 48 h. Cells were subsequently lysed with 25 μL of 1× passive lysis buffer (Promega) for 30 min at room temperature. Luciferase activity was measured on a Victor 3 multilabel plate reader (PerkinElmer) immediately after the addition of 25 μL of luciferin (Promega). All readouts were normalized to controls with no serum treatment. The ssCBA-Luc AAV was only used for the neutralizing antibody screen and not for in vivo delivery.
6
Live-cell Imaging with CellAsic System
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Live-cell imaging was performed exactly as described in King et. al. (2019) , except fresh SPO was used in place of conditioned SPO. Specific imaging conditions are noted in supplemental table 4. All time-lapse experiments were performed using the CellAsic system (EMD Millipore) in Y04D or Y04E microfluidics plates, with the exception of the Latrunculin A experiments, for which we used glass-bottom 96-well plates (Corning).
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