3D-MCTS were established by the hanging-drop method as previously described [22 (link)]. Briefly, 5,000 GL261 cells (National Cancer Institute, Frederick, MD) were suspended in 20 μL of spheroid media, prepared as described in the Online Resource file , on a lid of a petri-dish. The 3D-MCTS were then grown upside down by covering a phosphate buffered saline (PBS)-filled/moisturized petri-dish with the cell-seeded lid for 2 days. We then transferred the 3D-MCTS onto a sterile U-bottom 96-well plate (CELLSTAR®; Sigma-Aldrich, St. Louis, MO) and added RPMI 1640 medium (ThermoFisher Scientific, Waltham, MA). The spheroids were then incubated at 37 °C for 48 hours to reach ~500 μm in diameters prior to use. Subsequently, 3D-MCTS were treated with various BPN formulations carrying plasmids encoding a green fluorescent reporter protein, ZsGreen, at 1 μg DNA/spheroid. After 48 hours of incubation, the 3D-MCTS were transferred onto poly-D-lysine coated 35 mm glass bottom dishes (MatTek Corp., Ashland, MA). We then captured Z-stack fluorescence images at a depth of ~200 μm using an LSM 710 confocal microscope (Carl Zeiss; Hertfordshire, UK) under 10X magnification and conducted a quantitative analysis of spheroid area-normalized mean fluorescence intensity using an ImageJ software (NIH, Bethesda, MD).
Poly d lysine coated 35 mm glass bottom dishes
Manufactured by MatTek
Sourced in United States
Poly-D-lysine coated 35 mm glass bottom dishes are a type of laboratory equipment. They feature a glass bottom that is coated with the amino acid polymer poly-D-lysine, which promotes cell adhesion.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Cellstar, by Merck Group (1 mentions)
Fluorobrite dmem, by Thermo Fisher Scientific (1 mentions)
Fluoview fv1000 confocal microscope, by Olympus (1 mentions)
X tremegene 9 dna transfection reagent, by Roche (1 mentions)
Fluoview fv3000 confocal laser scanning microscope, by Olympus (1 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
T75 flask, by Corning (1 mentions)
510 meta confocal microscope, by Zeiss (1 mentions)
7 protocols using poly d lysine coated 35 mm glass bottom dishes
1
3D Tumor Spheroid Transfection
2
Fluorescent Protein Labeling in HEK293T Cells
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The HEK293T cells were
seeded at 25% confluency 24 h prior to transfection in poly-d -lysine-coated 35 mm glass bottom dishes (MatTek Corp.). Cells were
grown in DMEM supplemented with 10% FBS and penicillin-streptomycin
antibiotics until they reached 50–60% confluency. Transfections
of the plasmid encoding the SOD1-Halo or TDP43-Halo conjugate were
performed using X-tremeGene 9 DNA transfection reagent (Roche) according
to the manufacturer’s instructions. Proteins were expressed
for 24 h prior to analyses.
For confocal fluorescence imaging,
DMEM was replaced with FluoroBrite DMEM (ThermoFisher) supplemented
with 10% FBS, and Hoechst 33342 (0.1 μg/mL), and P9 (2.5 μM)
or TMR ligand (2.5 μM). The samples were incubated for 30 min
prior to imaging. To wash off unbound TMR ligands, the cells were
washed extensively by replacement of the medium with fresh DMEM and
incubation for 30 min at 37 °C. The medium was replaced with
fresh FluoroBrite DMEM (ThermoFisher) supplemented with 10% FBS prior
to imaging for the TMR-washed sample. Confocal images were obtained
using an Olympus FluoView FV1000 confocal microscope. The Halo–P9
conjugate fluorescence was visualized using a blue argon (488 nm)
laser. Nuclear staining was visualized using a violet laser (405 nm).
The TMR ligand was visualized using a green HeNe laser (543 nm).
seeded at 25% confluency 24 h prior to transfection in poly-
grown in DMEM supplemented with 10% FBS and penicillin-streptomycin
antibiotics until they reached 50–60% confluency. Transfections
of the plasmid encoding the SOD1-Halo or TDP43-Halo conjugate were
performed using X-tremeGene 9 DNA transfection reagent (Roche) according
to the manufacturer’s instructions. Proteins were expressed
for 24 h prior to analyses.
For confocal fluorescence imaging,
DMEM was replaced with FluoroBrite DMEM (ThermoFisher) supplemented
with 10% FBS, and Hoechst 33342 (0.1 μg/mL), and P9 (2.5 μM)
or TMR ligand (2.5 μM). The samples were incubated for 30 min
prior to imaging. To wash off unbound TMR ligands, the cells were
washed extensively by replacement of the medium with fresh DMEM and
incubation for 30 min at 37 °C. The medium was replaced with
fresh FluoroBrite DMEM (ThermoFisher) supplemented with 10% FBS prior
to imaging for the TMR-washed sample. Confocal images were obtained
using an Olympus FluoView FV1000 confocal microscope. The Halo–P9
conjugate fluorescence was visualized using a blue argon (488 nm)
laser. Nuclear staining was visualized using a violet laser (405 nm).
The TMR ligand was visualized using a green HeNe laser (543 nm).
3
3T3-L1 Adipocyte Imaging Protocol
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3T3-L1 pre-adipocyte cells were seeded in poly-D-lysine coated 3.5 mM glass bottom dishes (MatTek Corporation, Ashland, MA, USA) and differentiated as described above before dosing.
Cells were dosed as described above before imaging. Images were obtained at 37 °C in 5% carbon dioxide atmosphere on an Olympus FluoView FV3000 Confocal Laser Scanning Microscope (Olympus, Melbourne, Australia), equipped with an Olympus 60X water objective (UPLSAPO60XW) and 405, 488, and 561 nm lasers. Emission was collected for each of the lasers from 450 to 550 nm, 500–600 nm, and 570–670 nm, respectively.
Images were processed using FIJI software [32 (link)].
Cells were dosed as described above before imaging. Images were obtained at 37 °C in 5% carbon dioxide atmosphere on an Olympus FluoView FV3000 Confocal Laser Scanning Microscope (Olympus, Melbourne, Australia), equipped with an Olympus 60X water objective (UPLSAPO60XW) and 405, 488, and 561 nm lasers. Emission was collected for each of the lasers from 450 to 550 nm, 500–600 nm, and 570–670 nm, respectively.
Images were processed using FIJI software [32 (link)].
4
Cell Surface Receptor Binding Assay
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Cells grown in T-75 flasks (Corning) to 70-80% confluence were harvested with 10 mM EDTA in PBS for 15 min at 37 °C. Cells were washed twice with Iscove's Modified Dulbecco's Medium (IMDM, Gibco) supplemented with 10% FBS by centrifugation at 1,000 × g, for 2 min and resuspended in IMDM/FBS. Increasing ratios (multiplicities of incubation ranging from 20 to 150) of SSHEL αHER2 (labeled with AlexaFluor647) were incubated with 5 x 10 5 cells/ml in siliconized tubes (G-tubes, Bio Plas Inc.) for 1 h at 37 °C with gentle inversion. Cells were then washed with PBS once, resuspended in 1 ml PBS, and examined by flow cytometry (FACSCanto II, BD Biosciences). All data analysis was performed using FlowJo software (Tree Star). For confocal live cell imaging, 2.5 × 10 5 cells were seeded in poly-d-lysine-coated 35 mm glass-bottom dishes (MatTek) in 2 ml complete growth medium. After 24 h, cells were co-incubated with 5 x 10 7 SSHELs αHER2 labeled with AlexaFluor488 for 1 h at 37 °C. Cells were then washed twice with PBS, once with OptiMEM (Gibco), and stained with Hoechst 33342 (1 μg/ml in OptiMEM) for 30 min at 37 °C. Samples were washed three times with PBS and visualized using confocal microscopy (Zeiss LSM780).
5
Calcium Imaging of Astrocytes and HEK293 Cells
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Mixed cultures or HEK293 cells were grown in poly-D-Lysine coated 35 mm glass bottom dishes (MatTek) and transfected with plasmids containing either the Ca 2+ indicator GCaMP6f driven by the GFAP promoter (for astrocytes) or Lck-GCaMP6m driven by the CMV promoter (for HEK293 cells). Lck tag was added to promote the sensor recruitment to the plasma membrane, increasing the sensitivity of the detection in HEK293 cells. HEK293 cells were also co-transfected with a construct containing Cherry-GLT-1 in pCDNA3. Two days after transfection, the cells in the imaging chamber were challenged for 5 min with L-glutamate (1 mM) in aCSF (10 mM HEPES, 140 mM NaCl, 2.4 mM KCl, 2 mM CaCl 2, 1 mM MgCl 2 , 10 mM glucose [pH7.4]) at 37 °C. For GCaMP imaging, a single optical section was visualized for 5 min at a frequency of 0.2 Hz on a Zeiss AxioVert 200 M, 40Plan Neofluar NA 1.3 objective equipped with an ORCA-Flash4.0 LT CMOS camera. All images were processed and analyzed using FIJI/ImageJ software (Schindelin et al., 2012) , background correcting all images. Three ROIs were placed at different membrane regions of the cells to analyze GCaMP6 fluorescence. The fluorescence time course of each cell was measured by averaging all the pixels within the ROIs.
6
Live-Cell Imaging of Cellular Dynamics
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Cells were seeded onto poly-d -lysine–coated glass-bottom 35-mm dishes (MatTek Corp.), with media and transfection conditions as described above. Spinning disk and laser scanning confocal live-cell imaging was performed under environmentally controlled conditions, at 37°C and 5% CO2. For experiments using the spinning disk microscope, the protocol is as described in Stehbens et al. (2014 (link)) except that the system was upgraded with a next-generation scientific CCD camera (cMyo, 293 Photometrics) with 4.5-µm pixels, allowing optimal spatial sampling using a 60× NA 1.49 objective (CFI 294 APO TIRF; Nikon). For experiments using the Olympus Fluoview 1000 laser scanning confocal microscope, a 60× oil-immersion objective with NA 1.42 was used to obtain confocal images (1,024 × 1,024 pixels). Z-stack images were acquired with a step size of 0.5–1 μm and processed using the Fiji software package.
7
FRAP Analysis of GFP-TBP Dynamics
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