HepG2 cells were plated at 3000 cells/well in a 384 well plate (Corning) in growth medium. After an overnight incubation 10 μL of 5x compound was added and the plates were incubated overnight. The cells were then washed with phenol red free DMEM (Invitrogen) and incubated with 30 μl of BODIPY-FL-LDL (Invitrogen) diluted to final concentration 5 μg protein/mL in phenol red free DMEM for 0.1 to 5 hr. LDL uptake was stopped by fixing the cells with 3% formaldehyde. Nuclei were stained with Hoechst 33342 diluted in PBS for 10 minutes. The cells were then washed with PBS and imaged using an IXM microscope. Image analysis and quantification was performed with MetaXpress software (ver. 3.1.0.97, Molecular Devices) using the Multi Wavelength Cell Scoring application module.
Phenol red free dmem
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Phenol red-free DMEM is a cell culture medium that does not contain the pH indicator phenol red. It is a basal medium that provides essential nutrients for the growth and maintenance of cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (30 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (23 mentions)
Streptomycin, by Thermo Fisher Scientific (16 mentions)
Penicillin, by Thermo Fisher Scientific (15 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (13 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (11 mentions)
L glutamine, by Thermo Fisher Scientific (10 mentions)
Glutamax, by Thermo Fisher Scientific (8 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (8 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (7 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (6 mentions)
Insulin, by Merck Group (6 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (5 mentions)
L glutamine, by Merck Group (4 mentions)
Trypsin edta, by Thermo Fisher Scientific (4 mentions)
Charcoal dextran stripped fbs, by Thermo Fisher Scientific (3 mentions)
Tamoxifen, by Merck Group (3 mentions)
17β estradiol e2, by Merck Group (3 mentions)
Charcoal stripped fbs, by Merck Group (3 mentions)
Opti mem, by Thermo Fisher Scientific (3 mentions)
207 protocols using phenol red free dmem
1
Quantifying LDL Uptake in HepG2 Cells
2
Live Cell Imaging of Transfected Cells
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U2OS cells and C8-D1A astrocytes were cultivated in Dulbecco’s modified Eagle medium (DMEM) and Ptk2 cells were cultivated in Eagle's minimum essential medium (MEM). Both DMEM and MEM were supplemented with 10% (vol/vol) FBS, 100 units/mL penicillin, 100 μg/mL streptomycin and cells maintained at 37 °C and 5% (vol/vol) CO2 in a humidified incubator.
For transfection, cells were seeded on coverslips in a six-well plate. After one day cells were transfected with Lipofectamine LTX Reagent with PLUS reagent (Life Technologies, Carlsbad, USA) according to the manufacturer’s instructions. 24–48 h after transfection cells were washed and mounted with phenol red-free DMEM (Invitrogen, Carlsbad, USA) onto concavity slides. To prevent samples from drying, the coverslips were sealed with twinsil (Picodent, Wipperfürth, Germany). Finally, the cells were imaged at room temperature. Cells were mounted in phenol red-free DMEM (Invitrogen) and imaged 24–72 h post transfection.
For transfection, cells were seeded on coverslips in a six-well plate. After one day cells were transfected with Lipofectamine LTX Reagent with PLUS reagent (Life Technologies, Carlsbad, USA) according to the manufacturer’s instructions. 24–48 h after transfection cells were washed and mounted with phenol red-free DMEM (Invitrogen, Carlsbad, USA) onto concavity slides. To prevent samples from drying, the coverslips were sealed with twinsil (Picodent, Wipperfürth, Germany). Finally, the cells were imaged at room temperature. Cells were mounted in phenol red-free DMEM (Invitrogen) and imaged 24–72 h post transfection.
3
Live-cell and fixed-cell imaging of HeLa cells
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HeLa cells were plated on poly-lysine coated glass-bottom
dishes (MatTek, P35GC-1.5–14C), transfected, and treated as
described for in-gel fluorescence with the following modifications.
Following fluorophore incubation in culture media, cells were washed
3 × 1 mL PBS with the appropriate quench reagent, and quenched
for an additional 5 min in media. Cells were washed 3 × 1 mL
media to remove quench reagent and incubated in cell culture media
for 1–2 h prior to imaging. Cells were washed one time in phenol
red-free DMEM (Life Technologies)/10% FBS, and media was replaced
with phenol-red free DMEM/10% FBS supplemented with 10 μg/mL
Hoescht 33342 (Life Technologies, H3570) for nuclear labeling 5 min
prior to imaging live. For fixed-cell imaging, cells were fixed in
ice-cold MeOH for 10 min, washed 3 × 1 mL PBS and incubated in
1 mL PBS overnight at 4 °C. PBS was aspirated and 100 μL
VECTASHIELD (Vector Laboratories) cell mounting media containing DAPI
(Vector Laboratories, H-1200) was added prior to imaging.
dishes (MatTek, P35GC-1.5–14C), transfected, and treated as
described for in-gel fluorescence with the following modifications.
Following fluorophore incubation in culture media, cells were washed
3 × 1 mL PBS with the appropriate quench reagent, and quenched
for an additional 5 min in media. Cells were washed 3 × 1 mL
media to remove quench reagent and incubated in cell culture media
for 1–2 h prior to imaging. Cells were washed one time in phenol
red-free DMEM (Life Technologies)/10% FBS, and media was replaced
with phenol-red free DMEM/10% FBS supplemented with 10 μg/mL
Hoescht 33342 (Life Technologies, H3570) for nuclear labeling 5 min
prior to imaging live. For fixed-cell imaging, cells were fixed in
ice-cold MeOH for 10 min, washed 3 × 1 mL PBS and incubated in
1 mL PBS overnight at 4 °C. PBS was aspirated and 100 μL
VECTASHIELD (Vector Laboratories) cell mounting media containing DAPI
(Vector Laboratories, H-1200) was added prior to imaging.
4
Immunofluorescence Staining Protocol
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For immunofluorescence, the cells were cultured on glass coverslips or on gridded dishes (μ-Dish 35 mm Grid-500, Ibidi). The cells were fixed for 10 min in 4% PFA/PBS, quenched for 10 min in 50 mM NH4Cl/PBS and then permeabilized for 10 min with 0.1% Triton X-100/PBS. The cells were blocked in 2% BSA, 2% FBS/PBS for 30 min. Primary and secondary antibodies were diluted in blocking solution diluted 1:1 in PBS. The primary antibodies were incubated for a minimum of 1 h at RT, followed by washes in PBS; secondary antibodies were incubated for a minimum of 30 min at RT followed by washes in PBS. Coverslips were mounted with FluorSave (Millipore). For immunostaining against phosphorylated proteins, fixing solution was supplemented with PhosSTOP (1 tablet per 10 ml, Sigma), all PBS solutions were substituted with TBS, and blocking solution was substituted with 5% BSA/TBS. For surface immunostaining, the cells were washed in ice-cold phenol red-free DMEM (Gibco) and incubated with primary antibody diluted in ice-cold phenol red-free DMEM at 4 °C for 45 min. The cells were then washed in ice-cold PBS before fixation at RT in 4% PFA/PBS for 10 min. Secondary antibody staining was then carried out as outlined previously.
5
FRET Biosensor Imaging in Cell Culture
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5555‐EKAREV‐NLS were seeded onto a glass bottom 96‐well plate and cultured in 100 μl phenol red‐free DMEM (GIBCO/Thermo Fisher Scientific, Waltham, MA, USA) or mixture of 50 μl phenol red‐free DMEM and 50 μl rat CSF. For the dual‐emission ratio imaging, we used Zeiss 780 inverted microscope with 20× dry objective (Nikon Instruments Europe B.V., Amsterdam, Netherlands). The FRET biosensor was excited by a Chameleon Ti: Sapphire Laser (Coherent Inc., Cambridgeshire, UK) at 820 nm excitation wavelength through IR cut filter (MBC 760). The emission light was separated by beam splitters into 463–506 nm for CFP and 515–559 nm for YFP/FRET. Images were acquired as 12‐bit images and analysed with metamorph software (Universal Imaging), as described previously (Hirata et al., 2012 ).
6
Dextran-FITC Permeability Assay
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Transfected BMVEC were plated on tissue culture inserts with 3μm pores (Corning, Corning, NY) at 4 ×104 cells per insert and grown to confluence for 3–4 days. Cultures were maintained in puromycin containing media. Inserts were washed with phenol red-free DMEM (Thermo Fisher Scientific) and placed in 24-well tissue culture plates containing 400µL of phenol red-free DMEM/10% FBS in each well. Dextran-FITC (125 μg/mL, 70kDa, Sigma) (200µL) was added to the top of the insert, and after 5 min at 37°C, media was collected from the lower chamber and fluorescence was analyzed with a fluorescence-detecting plate reader (excitation λ 488 nm; emission λ 510 nm).
7
Inducible BANF1 Expression in iSLK Cells
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iSLK.219 cells in phenol red-free DMEM (Thermo Fisher) containing 10% Tet-free FBS (Clontech) and 1% l -glutamine were plated 24 h prior to the addition of transfection mixture containing 6 µg X-tremeGENE 9 DNA transfection reagent (Sigma) and 2 µg pCMV6-BANF1-HA in Opti-MEM. After 48 h, the media was replaced with phenol red-free DMEM (Thermo Fisher) containing 10% Tet-free FBS (Clontech), 1% l -glutamine, and 25 ng/mL doxycycline. Cells and supernatants were harvested at various timepoints post-reactivation for downstream analysis.
8
Dextran-FITC Permeability Assay
9
Estrogen-Dependent PTHrP Secretion in ER+ Tumor Cells
10
HeLa Cell Viability Assay
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HeLa cells were seeded in quadruplet into 96-well plates (Corning, Birmingham, UK) at 5 × 103 cells per well and incubated/beadfected as described. After incubation at 37 °C/5% CO2 for 50 h, cells were washed (three 100-μl washes) with phenol red-free DMEM (Invitrogen) supplemented with FBS, 100 IU/ml penicillin, 100 μg/ml streptomycin, 1 mm sodium pyruvate, 1× nonessential amino acid supplement, and 4 mm l -glutamine. The media was then replaced with fresh phenol red-free medium containing CellTiter-Blue® reagent (100 μl of medium + 20 μl of reagent) per well. Cells were incubated for a further 3 h at 37 °C/5% CO2, and then the absorbance at 620 nm was measured. After background subtraction, absorbance values were used to calculate the percentage viability, expressed as a percentage of untreated controls (which were set as 100% viable). The viability of cells beadfected with microspheres was compared with that of controls by means of one-way analysis of variance with Dunnett's multiple comparison test, with a q value less than 0.05 considered significant.
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