NIH/3T3 cells were plated at a density of 50,000 cells/ml in a glass-bottomed 24 well plate (MatTek, MA, USA) the day before transfection with plasmids encoding GFP, PDPN-CFP or PDPN-V5-his using Effectene transfection reagent (Qiagen, Hilden, Germany) as per supplier’s instructions. After 24 hours, cells were treated with chemical inhibitors at the concentrations indicated (see Extended data Fig. 1b ) for 6 hours before fixation. CLEC-2-Fc was added at 10 μg/ml for the time indicated in figures before fixation. In cotransfection experiments, plasmids encoding PDPN-CFP and ezrin-Cherry or PDPN-V5-his and CD44-GFP were added in equal amounts to the transfection mix. The cells were analysed by fluorescence microscope (20×, Nikon Eclipse Te2000-S, Tokyo, Japan) and contraction status scored manually. Cells were grouped into either contracted, partially contracted, spread or collapsed, depending on their morphology. High-resolution images were taken using a confocal microscope (Zeiss 710 using a 20×/0.8NA objective).
Glass bottomed 24 well plate
Manufactured by MatTek
Sourced in United States
The Glass-bottomed 24 well plate is a standard laboratory tool designed for cell culture and imaging applications. It features a transparent glass bottom that allows for high-quality microscopic observation and analysis of cells or other samples within the individual wells.
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3 protocols using glass bottomed 24 well plate
1
Analyzing Cell Morphology Dynamics
2
Cell Motility Imaging and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Cells were cultured in wells of a glass-bottomed 24-well plate (MatTek) in DMEM medium containing 10% FBS. Before imaging, the medium was changed to L-15 (Gibco) containing 10% FBS. Cell motility imaging was performed on an IX71 inverted Olympus microscope at 20× using multifield acquisition driven by MetaMorph software. Environmental conditions were controlled for the entirety of the 4-h motility experiment through the use of a heated chamber. Motility analysis was performed on images at 2-min intervals over the 4-h period as a way of fully analyzing a path of cell movement. Tracking was performed in ImageJ using the Manual Tracking and Chemotaxis Tool plug-ins freely available on the ImageJ website. A detailed explanation of motility parameters has been given previously (Soil, 1995 (link)). Centroid coordinates were user determined and later used to calculate the motility statistics presented in the results.
3
Analyzing Cell Morphology Dynamics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NIH/3T3 cells were plated at a density of 50,000 cells/ml in a glass-bottomed 24 well plate (MatTek, MA, USA) the day before transfection with plasmids encoding GFP, PDPN-CFP or PDPN-V5-his using Effectene transfection reagent (Qiagen, Hilden, Germany) as per supplier’s instructions. After 24 hours, cells were treated with chemical inhibitors at the concentrations indicated (see Extended data Fig. 1b ) for 6 hours before fixation. CLEC-2-Fc was added at 10 μg/ml for the time indicated in figures before fixation. In cotransfection experiments, plasmids encoding PDPN-CFP and ezrin-Cherry or PDPN-V5-his and CD44-GFP were added in equal amounts to the transfection mix. The cells were analysed by fluorescence microscope (20×, Nikon Eclipse Te2000-S, Tokyo, Japan) and contraction status scored manually. Cells were grouped into either contracted, partially contracted, spread or collapsed, depending on their morphology. High-resolution images were taken using a confocal microscope (Zeiss 710 using a 20×/0.8NA objective).
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