Parental HT-1080, HT-BFP, HT-HER2-GFP, and SKBR3 cells were plated overnight at 50% confluency on 96-well plates (Ibidi). Tzm-A647 was incubated with the cells for 30 minutes at 5% CO2 and 37 °C at 5 μg/mL. The cells were then washed 5 times with PBS, and fixed with 4% PFA (EMS). The cells were imaged with a DeltaVision (Applied Precision) modified BX63 microscope (Olympus) fitted with a camera (Andor).
96 well plate
Manufactured by Ibidi
Sourced in Germany
The 96-well plates are a common laboratory equipment used for various purposes in scientific research, such as cell culture, enzymatic assays, and drug screening. These plates consist of a grid of 96 individual wells, each with a defined volume capacity, designed to accommodate small sample volumes. The 96-well plate format allows for efficient, high-throughput experimentation and data collection.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (5 mentions)
Matrigel, by BD (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
In cell analyzer 2200 system, by GE Healthcare (2 mentions)
Matrigel, by Ibidi (2 mentions)
Alexa flour 488, by Thermo Fisher Scientific (1 mentions)
In cell analyzer 2000, by GE Healthcare (1 mentions)
Alexa 647, by Thermo Fisher Scientific (1 mentions)
Cyanine3 (cy3), by Jackson ImmunoResearch (1 mentions)
Phase contrast microscope, by Olympus (1 mentions)
Dmi6000b microscope, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Murine m csf, by Thermo Fisher Scientific (1 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Bio-Techne (1 mentions)
Sirna transfection reagent, by Horizon Discovery (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
32 protocols using 96 well plate
1
Imaging Membrane Protein Localization
2
Endothelial Cell Migration Assay
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Endothelial cells were seeded on 96‐well plates (Ibidi, Martinsried, Germany) coated with fibronectin. Once confluent, a strip of cells was removed using a sterile 96‐well wound‐making device (Essen Bioscience, Ann Arbor, MI, USA) and migration followed for up to 24 hours by live cell imaging (IncuCyte). Percentage of wound closure was analysed using the IncuCyte 2010A software. Representative pictures in the results section show an overlay of the initial cell layer immediately after wounding (black area) and the endothelial cell layer after 24 hours (grey area) for each group.
3
Quantifying Cell Cycle Dynamics
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Cells were plated into 96-well plates (Ibidi). Cells in the S phase were labeled using the Clik-It EdU Alexa Fluor 488 imaging kit (Life Technologies). Cells were washed twice with PBS then treated with hypotonic buffer (10 mM Tris-HCl, pH 7.4, 2.5 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, and 0.5% NP40) for eight min on ice. Cells were fixed with 4% paraformaldehyde in PBS for 15 min, and then treated with 0.5% Triton X-100 in PBS for 30min. Cells were stained with a specific primary Ab, then stained with species-specific Cy3 (Jackson ImmunoResearch Laboratories), Alexa Flour 488 (Life Technologies) or Alexa 647 (Life Technologies) secondary Abs. Nuclei were labeled with Hoechst 33258. Fluorescence images were obtained using an automated fluorescence microscope and IN Cell Analyzer 2000 (GE Healthcare BioScience). Cell cycle distribution was determined by the intensity of Hoechst 33258 and EdU labeling. The number of foci per cell at each cell cycle phase were determined using the image-analysis software provided with the IN Cell Developer (GE Healthcare BioScience).
4
Endothelial Tube Formation Assay
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Matrigel (10 μL; BD) was added to 96-well plates (ibidi, Germany) and incubated at 37°C for 30 min. Then, treated cells (2 ×104/well) resuspended in 50 μL of complete EGM-2 were seeded on the Matrigel and incubated at 37°C. Tube formation was examined 6 h later. The digital images of endothelial tubes were photographed with a phase-contrast microscope (Olympus, Japan).
5
Quantifying MCMV Infection in MEF Cells
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MEF were seeded in 96-well plates (Ibidi, Germany) at a density of 4x104 per well. 24 h later cells were infected with 2000 pfu/well of MCMV C3X-gfp using centrifugal enhancement (5min, 500xg) to enable synchronous infection. 4h later the inoculum was removed and cells were incubated in 300 μl/well medium containing 20 μg/ml mAb or serum at a dilution of 1:100. Infection was documented using a Leica DMI 6000B microscope starting at day 3 post infection. Magnification: 200fold. Filter: excitation 488 nm, emission 509, exposure times: 40 ms, picture size: 640 μm x 478 μm.
6
Cellular Uptake of Fluorescent Probes
7
Macrophage Characterization with AXL1717 and Anti-IGF1R
8
Irradiation Effects on Fibroblast Viability
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As described elsewhere (18) , irradiations with 350 MeV/n 40 Ar (104 keV/lm LET) and 600 MeV/n 56 Fe (170 keV/lm LET) were performed at Brookhaven National Laboratory (BNL; Upton, NY), while X-ray irradiation was performed at Lawrence Berkeley National Laboratory (LBNL) using the 160-kVp Faxitront X-ray machine (Lincolnshire, IL). In addition to the nonirradiated control, we studied two irradiation fluences for high-LET irradiation (1.1 and 3 particles/ 100 lm 2 , which correspond to doses of 0.18 and 0.50 Gy for 40 Ar, and 0.30 and 0.82 Gy for 56 Fe, respectively) and three X-ray doses (0.1, 1 and 4 Gy). The dose rate was 1 Gy/min for all conditions. Each condition was duplicated.
Fibroblasts were seeded at 10 4 cells/well in 96-well plates (IBIDI, Planegg, Germany) and incubated at 378C, 5% CO 2 and 3% O 2 for 4 days before irradiation. Media was replaced at 2 h prior to irradiation. After irradiation, fibroblasts were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) at 4, 24 and 48 h postirradiation (with an additional time point at 8 h for high-LET irradiation). Plates were sealed and stored at 48C until imaging.
Fibroblasts were seeded at 10 4 cells/well in 96-well plates (IBIDI, Planegg, Germany) and incubated at 378C, 5% CO 2 and 3% O 2 for 4 days before irradiation. Media was replaced at 2 h prior to irradiation. After irradiation, fibroblasts were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) at 4, 24 and 48 h postirradiation (with an additional time point at 8 h for high-LET irradiation). Plates were sealed and stored at 48C until imaging.
9
Lymphangiogenic Potential of pRCC Cells
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The pRCC cells were previously cultured for 24 h. The conditioned medium was then collected from different groups. HLECs were resuspended in collected conditioned medium and cultured for 12h in 96-well plates (IBIDI, German) placed with 10 μl Matrigel (BD Biosciences, USA). Using inverted fluorescence microscopy to observe tube formation, the formed lymphatic vessels were analyzed and counted by Image J software (NIH, USA).
10
Mapping Extracellular Matrix-Mediated Invadopodia
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MEMAs were produced as described previously (Smith et al., 2019; Watson et al., 2018) with several minor modifications. The same set of 45 ECM or ECM combinations that we previously reported were plated into the bottom of 96 well plates (Ibidi). The plates were left desiccated for several days at room temperature, then 3000 MDA-MB-231 cells containing Tks5aGFP were plated in 100 µl DMEM containing 10% FBS. After overnight adhesion in a 37 °C incubator with 5% CO2, an additional 100 µl of DMEM containing PBS control, epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2), or hepatocyte growth factor (HGF) was added to give final concentrations of 10 ng/ml EGF and FGF2 and 40 ng/ml of HGF. The cells were left to grow for 24h, then fixed with 4% PFA and washed with PBS three times. The cells on MEMA were stained with Alexa Fluor-568-phalloidin to visualize the invadopodia, which were also marked with Tks5aGFP, where double positive delineated invadopodia). Hoechst was used for staining of cell nuclei. Imaging was performed on laser-scanning confocal microscope LSM880 equipped with AiryScan.
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