A modified transwell system was used to measure the uptake of HBPE-NPs by cancer cells after interactions with endothelial cells [19 (link)]. Briefly, a Millicell- 24 cell culture insert plate (MilliporeSigma, Burlington, MA, USA) was seeded with 3 × 104 HUVEC cells that were 80% confluent after 48 h. The insert was transferred to a 24-well glass-bottom plate (Cellvis, Mountain View, CA, USA), previously seeded with 5 × 104 MDA-MB-231 cells grown to 60% confluency. To the insert with HUVEC cells, 0.1 mg of PEGylated or sera-treated HBPE-NPs was added. After 24 h, to assess the uptake of HBPE-NPs, MDA-MB-231 cells were fixed for imaging individual cells (LSM 710 confocal microscope) or total fluorescence (Cytation 5 multi-modal plate reader). Confocal images were taken at a 20× (MDA-MB-231, THP-1) or 10× magnification (HUVEC). Cytation images were taken at a 10× magnification. Fluorescence was analyzed with ZEN Blue software (Carl Zeiss AG, Dublin, CA, USA).
24 well glass bottom plate
Manufactured by Cellvis
Sourced in United States
The 24-well glass-bottom plate is a laboratory equipment item designed to provide a transparent surface for cell culture and microscopy applications. Each well in the plate features a thin glass bottom, allowing for clear optical access to the cells or samples being observed or imaged.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880 confocal microscope, by Zeiss (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Alexa fluor 488 conjugated goat anti rabbit secondary antibody, by Thermo Fisher Scientific (2 mentions)
800 airyscan microscope, by Zeiss (2 mentions)
Image express micro confocal high content imaging system, by Molecular Devices (2 mentions)
Alexa fluor 488 647 conjugated secondary antibodies, by Thermo Fisher Scientific (2 mentions)
Rabbit anti phospho upf1 antibody, by Merck Group (2 mentions)
Zen blue software, by Zeiss (2 mentions)
Turbofect, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Mitotracker green, by Thermo Fisher Scientific (2 mentions)
Fluospheres microspheres, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Lsm 780 inverted microscope, by Zeiss (2 mentions)
Doxycycline, by Merck Group (2 mentions)
Lionheart fx automated microscope, by Agilent Technologies (2 mentions)
Fugene hd, by Promega (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Millicell 24 cell culture insert plate, by Merck Group (1 mentions)
34 protocols using 24 well glass bottom plate
1
Transwell Assay for HBPE-NP Uptake
2
Imaging U2OS Cells During Cell Cycle
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U2OS cells were seeded into a 24-well glass-bottom plate (Cellvis) 48 h prior to imaging. The culture medium was replaced with phenol red-free medium 8–12 h prior to imaging. Time-lapse images were acquired with an automated inverted microscope Nikon Ti-E with a Plan-Apo 40×/0.85 objective (Nikon). The microscope was equipped with a white-light LED (Lumencor SOLA) and an sCMOS camera (Hamamatsu ORCA-Flash4.0V2). Standard fluorescence microscopy filter cubes (Chroma and Semrock) were used for the acquisition of CFP, GFP, YFP, mCherry and iRFP fluorescence. The sample stage was maintained under 37°C and 5% CO2 humidified air in a custom environmental chamber. Multi-channel fluorescence images were acquired every 10 min using a custom Micro-Manager (41 (link)) (v1.4) script for 24 to 48 h.
For imaging experiments with the cell line 3-B5-6/Citrine-PCNA, doxycycline was added 12 h prior to imaging to ensure steady-state transcription. The medium was replaced with fresh phenol red-free medium with doxycycline of the same concentration before imaging. For cell cycle blocking experiments, 2 mM thymidine (Sigma-Aldrich, T9250) was added after imaging. Images of mCherry, YPF, CFP and brightfield were captured every 10 min for 48 h. For imaging nascent transcription sites, four z-stacks images with 1.2 μm spacing were acquired to capture loci in different focal planes.
For imaging experiments with the cell line 3-B5-6/Citrine-PCNA, doxycycline was added 12 h prior to imaging to ensure steady-state transcription. The medium was replaced with fresh phenol red-free medium with doxycycline of the same concentration before imaging. For cell cycle blocking experiments, 2 mM thymidine (Sigma-Aldrich, T9250) was added after imaging. Images of mCherry, YPF, CFP and brightfield were captured every 10 min for 48 h. For imaging nascent transcription sites, four z-stacks images with 1.2 μm spacing were acquired to capture loci in different focal planes.
3
Live Cell Imaging of NIH3T3 Cells
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Cells were plated on 0.17±5 μm 24 well glass bottom plate (Cellvis) in the FluotoBrite™ DMEM (ThermoFisher A1896701) overnight. All live cell imaging was performed using 60X oil immersion objective (NA 1.4) on a Nikon Ti-Eclipse with a temperature stage at 37°C and supplied with 5% CO2. NIH3T3 cells were imaged using four wavelengths (405 nm for tagBFP; 488 nm for Citrine; 561 nm for mCherry or FusionRed; 674 nm for SiR700-Tubulin Kit, #CY-SC014, Cytoskeleton Inc.).
4
Calcium-Induced Dynamics in HeLa Cells
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HeLa cells (40% to 60% confluent) on 24-well glass bottom plate (Cellvis, Mountain View, California, USA) were transfected with 0.5 μg of plasmid DNA and 2 μl TurboFect (Thermo Fisher Scientific). Following 2-hour incubation, the media was changed to DEME (Gibco, Waltham, Massachusetts, USA) with 10% fetal bovine serum (FBS) (Sigma), 2 mM GlutaMax (Thermo Fisher Scientific), and 1% penicillin-streptomycin (Gibco), and the cells were incubated for 48 hours at 37°C in a CO2 incubator before imaging. Prior to imaging, culture medium was changed to Hank’s Balanced Salt Solution (HBSS). Wide-field imaging was performed on a Nikon Eclipse Ti microscope that was equipped with a 75 W Nikon xenon lamp, a 16-bit 512SC QuantEM EMCCD (Photometrics, Tucson, Arizona, USA), and a 60× objective and was driven by a NIS-Elements AR 4.20 software package (Nikon, Tokyo, Japan). For time-lapse imaging, HeLa cells were treated with 4 mM EGTA (with 5-μM ionomycin) and then 10 mM CaCl2 (with 5-μM ionomycin). Images were taken every 5 seconds using a filter set with 650/60-nm excitation and 720/60-nm emission.
5
Immunofluorescence Staining of Transfected Cells
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Cells grown on glass coverslips in 24-well plates were fixed for 10 min with 4% paraformaldehyde (PFA), permeabilized in permeabilization Buffer (0.3% Igepal CA-630, 0.05% Triton-X 100, 0.1% IgG-free BSA in PBS) for 3 min, then blocked in blocking buffer (0.05% Igepal CA-630, 0.05% Triton-X 100, 5% normal goat serum in PBS) for 60 min. Primary and secondary antibodies were applied in blocking buffer for 1 h. The nucleus was stained with Hoechst-33342 (sc-200908, Santa cruz Biotechnology, Dallas, TX, USA). Cells were washed three times with wash buffer (0.05% Igepal CA-630, 0.05% Triton-X 100, 0.2% IgG-free BSA in PBS) and twice with PBS. Coverslips were mounted using ProLong Gold Antifade Reagent (ThermoFisher).
For the cell surface staining of 3×Flag-KDELR1-mCherry, HeLa S3 cells were seeded on a 24-well glass-bottom plate (Cellvis) coated with fibronectin (Millipore, Burlington, MA, USA). After 18 h transfection with 3×Flag-KDELR1-mCherry and indicated plasmid, cells were incubated with anti-Flag antibody (Sigma) in DMEM with 2% FBS at 4 °C for 1 h. The cells were washed twice with ice-cold PBS, and then fixed using 4% PFA. After washing three times with PBS, the cells were incubated with secondary antibody for 1 h. Cells were washed three times and then were imaged with a 63× objective on a Zeiss LSM 880 confocal microscope.
For the cell surface staining of 3×Flag-KDELR1-mCherry, HeLa S3 cells were seeded on a 24-well glass-bottom plate (Cellvis) coated with fibronectin (Millipore, Burlington, MA, USA). After 18 h transfection with 3×Flag-KDELR1-mCherry and indicated plasmid, cells were incubated with anti-Flag antibody (Sigma) in DMEM with 2% FBS at 4 °C for 1 h. The cells were washed twice with ice-cold PBS, and then fixed using 4% PFA. After washing three times with PBS, the cells were incubated with secondary antibody for 1 h. Cells were washed three times and then were imaged with a 63× objective on a Zeiss LSM 880 confocal microscope.
6
Immunofluorescence Staining of HEK293T and iPSNs
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HEK293T cells were fixed and stained as previously described (Coyne et al., 2020 (link)) using Rabbit anti-phospho-UPF1 antibody (Millipore Sigma) at a 1:250 dilution and AlexaFluor 488 conjugated Goat anti-Rabbit secondary antibody (Thermo Fisher Scientific) at a 1:1000 dilution in a 24-well glass-bottom plate (Cellvis). 9 sites in each well were imaged using a 20x objective on an ImageExpress Micro Confocal High-Content Imaging System (Molecular Devices).
iPSNs were fixed with 4% (v/v) para-formaldehyde in PBS for 10 min, permeabilized in 0.2% (v/v) Triton X-100 for 10 min, blocked in 1% bovine serum albumin and 2% goat serum for 1h, incubated with primary antibodies overnight at 4°C, washed with PBS, and finally incubated with Alexa Fluor 488/647 conjugated secondary antibodies (ThermoFisher Scientific). Cells were imaged with a Zeiss 800 Airyscan microscope.
iPSNs were fixed with 4% (v/v) para-formaldehyde in PBS for 10 min, permeabilized in 0.2% (v/v) Triton X-100 for 10 min, blocked in 1% bovine serum albumin and 2% goat serum for 1h, incubated with primary antibodies overnight at 4°C, washed with PBS, and finally incubated with Alexa Fluor 488/647 conjugated secondary antibodies (ThermoFisher Scientific). Cells were imaged with a Zeiss 800 Airyscan microscope.
7
Visualizing Cell Cytoskeleton and Lipid Rafts
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Cells were seeded onto a 24-well glass bottom plate (Cellvis, Mountain View, CA) and fixed with 4% paraformaldehyde (PFA) for 1 h at room temperature. Cells were rinsed with PBS and incubated with 1 ml glycine (1.5 mg /ml PBS) for 10 min at room temperature to quench the PFA. Cells were stained with freshly prepared 50 μg/ml filipin III (Sigma) in PBS for 3 h at room temperature followed by Alexa Fluor 488 Phalloidin (Life Technologies/Molecular Probes, Eugene, OR; 1:50) in PBS. Stained cells were viewed on an Olympus Fluoview 500 confocal microscope (Olympus America, Inc., Center Valley, PA).
8
Immunofluorescence Staining of Adherent Cells
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Cells grown on a 24-well glass bottom plate (Cellvis) were fixed with 4% paraformaldehyde (PFA) for 10 min, permeabilizated in permeabilization buffer (0.05% Triton-X100 in PBS) for 3 min, and blocked in blocking buffer (3% BSA, 0.05% Triton-X100 in PBS) for 60 min. Then the cells were incubated with primary and secondary antibodies in blocking buffer for 1 h. The nucleus was stained with Hoechst-33342 Santa cruz Biotechnology). Cells were washed three times with wash buffer (0.2% BSA, 0.05% Triton-X100 in PBS) and twice with PBS. Images were acquired using a Zeiss LSM880 confocal microscope with a 63 × oil immersion objective.
9
Glucose Starvation Dynamics via Lyso-ExRai-AMPKAR
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Cells were grown on 24 well glass-bottom plate (Cellvis) and transfected with Lyso-ExRai-AMPKAR. After 20–24 h, cells were incubated with or without glucose-free medium for another 2 h. For glucose starvation, we used glucose-free DMEM (Gibco, 11966-025) supplemented with 10% FBS. Images were then acquired by Olympus FV-3000 confocal microscope (100× oil objective, NA = 1.45). X, Y scans were acquired at 8 μs/pixel with 1024 × 1024 resolution (sequential mode: line). Dual GFP excitation-ratio imaging was performed (405 nm excitation: PMT voltage = 618 V, 7% laser transmissivity, detection wavelength is 465–565 nm; 488 nm excitation: PMT voltage = 503 V, 3.8% laser transmissivity, detection wavelength is 465–565 nm), and images of each group were captured within 15 min. The fluorescence emission with 488 nm excitation and 405 nm excitation was measured using ImageJ software, and the ratio of these two fluorescence intensities (excitation 488/excitation 405) was calculated for further analysis.
10
Immunofluorescence Staining of HEK293T and iPSNs
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