HeLa cells were seeded out at a density 150,000 cells/mL in Fluorobrite DMEM media (Gibco) in T75 culture flasks (Thermo Fisher Scientific). One day later the cells were incubated with TNF-α (20 ng/mL, Sigma-Aldrich) at 37°C for 24 h. Cells were washed twice in PBS and incubated in ice-cold Fluorobrite DMEM media with Stx2:Alexa 488 (200 ng/mL, final concentration) for 1 h in 4°C. Cells were washed as above and incubated with A23187 10 μM for 40 min to induce microvesicle release. The cell media was collected and centrifuged at 2,500 × g for 5 min and the supernatant, containing microvesicles, was collected and washed as described above. These microvesicles were used for detection and quantification of microvesicle uptake by DLD-1 cells.
Fluorobrite dmem media
Manufactured by Thermo Fisher Scientific
Sourced in United States
FluoroBrite DMEM media is a cell culture media formulation developed by Thermo Fisher Scientific. It is designed to support the growth and maintenance of a variety of cell lines in in vitro cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (10 mentions)
Acumen, by SPT Labtech (4 mentions)
Prism 7, by GraphPad (3 mentions)
Imagexpress micro xl, by Molecular Devices (3 mentions)
Growth factor reduced matrigel, by Corning (3 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (2 mentions)
Incucyte zoom, by Sartorius (2 mentions)
Mt10013cv, by Corning (2 mentions)
Penn strep, by Corning (2 mentions)
2 beta mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (2 mentions)
L glutamine, by Corning (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (2 mentions)
Pen strep, by Thermo Fisher Scientific (2 mentions)
Fluo 4 am, by Thermo Fisher Scientific (2 mentions)
Ab175670, by Abcam (2 mentions)
53 protocols using fluorobrite dmem media
1
Microvesicle Isolation and Uptake Assay
2
Dual-Modality Imaging of COS-7 Cells
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COS-7 cells were seeded according to the procedure described previously. After 12–16 h cells were transfected with actinin-mEOS2 or Lifeact-mEOS2 (Supplementary Table 1 ) with a Lipofectamine 3000 (Thermo Fisher) according to the protocol provided by manufacturer. Cells were used for combined SICM-SOFI experiment 24 h after the transfection. Imaging was performed at 37 °C and 5% CO2 in a custom-built imaging chamber in FluoroBrite DMEM media (Gibco, Thermo Fisher Scientific) in order to reduce autofluorescence. SICM and SOFI imaging was performed subsequently i.e., after each SICM image, a fluorescent stack of 300 frames with an exposure time of 50 ms was recorded. The first 50 frames were excluded from SOFI processing due to rapid intensity change upon 405 activation and only 250 frames were used for further SOFI analysis. Live-cell imaging was performed under the low-illumination intensity (500 W/cm2 of 561 imaging laser and 0.3 W/cm2 of 405 nm activation laser) in order to reduce phototoxicity.
3
Mitochondrial Dynamics in MEF and HeLa Cells
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wt and Drp1-null mouse embryonic fibroblast (MEF) cells48 (link) and human cervical adenocarcinoma (HeLa S3) cells were grown in DMEM supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin at 37 °C in 5% CO2 atmosphere. Before imaging, the cells were plated at 50–60% confluency in glass-bottom microscopy dishes (35 mm, N° 1.5 glass, 14 mm well-diameter, MatTek, Ashland, MA) pre-coated with fibronectin/gelatin solution (10 μg/mL/20 μg/mL). Cells were transfected with 1–2 μg of plasmid DNA encoding either mEGFP alone or the mEGFP Drp1 fusion constructs together with the mCherry-Mito-7 plasmid (gift from Michael Davidson, Addgene plasmid #55102) using the Lipofectamine LTX kit (Invitrogen) according to the manufacturer’s protocol. Cells were incubated for 24–48 h post-transfection. One hour before imaging, the culture medium was exchanged with Fluorobrite DMEM media (Gibco) supplemented with 1X GlutaMAX and 10 mM HEPES, pH 7.2 (Gibco).
4
Quantifying Myoblast Differentiation via Imaging
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C2C12 cells under select experimental conditions were allowed to differentiation for 4 days before imaging. Cells were washed with 1×PBS and mitochondrial were stained by being exposed to 50 nM of MitoTracker® Red CMXRos (Molecular Probes™) in FluoroBrite™ DMEM Media (Gibco) for 30 min at 37 °C. Cells were then washed with 1×PBS and incubated with DAPI in FluoroBrite™ media for 20 min at room temperature. Cells were imaged by florescence at 10× magnification. Nuclei counting was conducted in ImageJ. Total differentiation was calculated by counting the number of nuclei within the myotubes divided by the total number of nuclei with in the image (N = 3). The average the level if myoblast fusion was calculated as follows: number of nuclei per myotube/total number of myotubes (N = 3).
5
E3 Ligase Binding Assay Protocol
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The E3 ligase in vitro binding assays were adapted from Nowak et al. (2018) (link). Briefly, a day before compound treatment, cells stably expressing BRD4BD2-GFP with mCherry reporter were seeded at 30–50% confluency in 384-well plates (3764, Corning) with 50 μL FluoroBrite DMEM media (Gibco, A18967) containing 10% FBS per well. Compounds and either 250 nM AT-1 (Gadd et al., 2017 (link)) for the VHL engagement assay or 100 nM dBET6 (Winter et al., 2017 (link)) for CRBN engagement assay, were dispensed using D300e Digital Dispenser (HP) normalized to 0.5% DMSO and incubated with cells for 5 h. The assay plate was imaged immediately using Acumen eX3/HCl (TTPLabtech) High Content Imager with 488 and 561 nm lasers in 2 × 1 μm grid per well format. The GFP/RFP ratio of the resulting images was analyzed using a CellProfiler pipeline (Carpenter et al., 2006 (link)). The GFP/mCherry ratio was normalized to DMSO and fitted in GraphPad Prism 7 using variable slope equation.
6
Visualizing Receptor Dynamics in Live Cells
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Prior live-cell imaging, 24 h post-transfection, Cos-7 or HUVEC cells grown on 25 mm #1.5H glass coverslips (Thorlabs, CG15XH1) were incubated for at least 30 min in serum-free FluoroBrite DMEM media (Gibco, A1896701) supplemented with 5 μM biliverdin (co-factor for iRFP713; Cayman Chemical, 1925710). Time-lapse TIRF imaging was performed following stimulation with hEGF (100 ng/ml21 (link); Millipore Sigma, E9644-.2MG) for Cos-7 cells, or recombinant hVEGF 165 (50 ng/ml60 (link); Fischer Scientific, 293VE010CF) for HUVECs. Each cell was imaged simultaneously with TIRF 488 and TIRF 647, for 5 min with 200 ms exposure time and 100 ms delay between frames. Three to four cells were imaged per ligand stimulation.
7
SARS-CoV-2 Infection Kinetics Assay
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Vero E6 or A549-ACE2 cells were seeded overnight in 24-well plates (Corning) at a density of 2x104 cells per well. After cytokine treatment and infection with SARS-CoV-2-mNG (MOI of 0.1 for Vero E6; or MOI of 1 for A549-ACE2), cells were washed twice and imaged in FluoroBrite DMEM media (Gibco) by live-cell fluorescence microscopy using the FITC filter set on a Leica DM IRB inverted modulation contrast microscope. Image acquisition was carried out with a FLUOTAR 10x objective controlled by Leica Microsystems Application Suite X software. For A549-ACE2 experiments, NucBlue nuclear staining for live cells (Invitrogen) was added (2 drops per mL of media) in the last 15 minutes of incubation and detected through a DAPI filter set. In some experiments, Vero E6 cells were infected with SARS-CoV-2 at a MOI of 1 to visualize the amount of virus required to produce CPE in 50% of inoculated tissue culture cells (TCID50 assay). Representative images of cytokine treated cells displaying viral CPE (in 24-well plates) were taken at 96 h post-infection (to match the time point where the 96-well plates containing cell supernatants were scored) with a HI PLAN PH1 10x objective on a Leica DMi1 inverted phase contrast digital microscope. Leica Microsystems Application Suite software was used for image acquisition.
8
Cardiomyocyte Differentiation of hiPSCs
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The hiPSC-lines used in the study were purchased from Riken Cell Bank (Ibaraki, Japan). Cells were cultured on iMatrix-511 (Takara Bio, Tokyo, Japan) coated dishes in StemFit AK02N medium (Ajinomoto, Tokyo, Japan) at 37°C and 5% CO2. The medium was changed daily. For cardiomyocyte differentiation, cells were cultured until 70% confluency and induced to differentiate using a PSC Cardiomyocyte Differentiation Kit (Gibco, Waltham, MA) following the manufacturer's instructions. For Raman measurements, cells were harvested using TrypLE Select (Gibco) and washed with PBS and centrifuged at 150×g for 5 min before being resuspended in FluoroBrite DMEM Media (Gibco).
9
Calcium Imaging of Cultured CAFs
10
Live imaging of mitochondrial dynamics
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For live imaging, cells were differentiated in 35 mm µ-dishes (81156, Ibidi Germany) at 3x10 4 cells/cm 2 and treated with 6-OHDA and rotenone. Subsequently, mitochondria were stained with 25 nM of the mitochondrial fluorescent dye MitoTracker Red CMXRos (M7512, Invitrogen, Thermo Fisher Scientific) in the FluoroBrite DMEM Media (A1896702, Gibco, Thermo Fisher Scientific) for 30 min. The media was then replaced by fresh FluoroBrite DMEM Media. Movies of fluorescent mitochondria were then recorded using the TIRF-fitted Nikon Eclipse Ti2 inverted microscope. The lowest level of excitation light from the 561 nm laser was used for imaging, and the emitted light was collected using an mCherry filter. The EMCCD Andor iXon Ultra DU888 camera (Andor Technologies) was used to capture the images with resolution of 1024 x 1024 pixels (pixel size 13 x 13 μm) at 1 frame per second for 10 min.
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