GRKO or Parental cell lines were plated in complete medium in two-well LabTek II chamber slides. The next day, HaloTag MR or H2B constructs were transfected using jetOPTIMUS (Polyplus) following manufacturer’s protocol. After incubation for 4 hours with the jetOPTIMUS reaction mix, the medium was replaced with DMEM medium supplemented with charcoal/dextran-stripped FBS. 24 hours later, cells were incubated for 20 min with 5 nM of the cell-permeant HaloTag ligand Janelia Fluor 646 (JF646). After labeling, cells were washed three times for 15 min with phenol red-free DMEM media (Gibco) supplemented with charcoal/dextran-stripped FBS, followed by one last wash after 10 min, to remove unbound JF646. Cells were then treated with 10 nM Aldo or 100 nM Cort for 30 min before imaging.
Phenol red free dmem media
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States
Phenol red-free DMEM media is a basal medium formulation that lacks the pH indicator phenol red. It is commonly used for cell culture applications where the presence of phenol red may interfere with certain experimental procedures or analyses.
Automatically generated - may contain errors
Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (4 mentions)
Blasticidin, by GoldBio (2 mentions)
Puromycin, by Thermo Fisher Scientific (2 mentions)
293fectin, by Thermo Fisher Scientific (2 mentions)
Kolliphor p188, by Merck Group (2 mentions)
Hek293 6e, by American Type Culture Collection (2 mentions)
Lipofecatime 3000, by Thermo Fisher Scientific (2 mentions)
Hek293 6e, by Merck Group (2 mentions)
F17 media, by Merck Group (2 mentions)
Hek293, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (2 mentions)
Jetoptimus, by Polyplus Transfection (1 mentions)
Lsm 980 elyra 7, by Zeiss (1 mentions)
Live cell imaging dishes, by Ibidi (1 mentions)
Glass bottom chamber slides, by Ibidi (1 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
Fugene hd transfection reagent, by Promega (1 mentions)
Hek293, by American Type Culture Collection (1 mentions)
Axioobserver z1, by Thermo Fisher Scientific (1 mentions)
11 protocols using phenol red free dmem media
1
Mineralocorticoid receptor live imaging
2
Stable Cell Line Generation for Biosensors
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Stable cell lines were generated using either human embryonic kidney (HEK) HEK293 (ATCC, Manassas, VA) or HEK293-6E (National Research Council, Canada) cells25 (link). Briefly, cells were transiently transfected with 2CS, 1CS, or G32R null-biosensor plasmids using Lipofecatime 3000 or 293fectin (Thermo Fisher Scientific). Flow cytometry was used to select and enrich for the population of cells expressing respective biosensors. Stable HEK293 cell lines were maintained in phenol red-free DMEM media (Gibco, Waltham, MA) supplemented with 2 mM GlutaMAX (Gibco, Waltham, MA), 10% fetal bovine serum (FBS) (Atlanta Biologicals, Lawrenceville, GA), 1 IU/mL penicillin/streptomycin (Gibco, Waltham, MA), and 250 μg/mL G418 (Fisher Scientific). Stable HEK293-6E cell lines were maintained in F17 media (Sigma Aldrich) supplemented with Kolliphor p188 (Sigma Aldrich, St. Louis, MO), 200 nM/mL GlutaMAX, and either 1 μg/mL puromycin (Invitrogen, Carlsbad, CA) or 2 μg/mL blasticidin (Goldbio) as a selection antibiotic. All cell lines were grown at 37 °C with 5% CO2.
3
Single-particle tracking of Rab7 and Arl8b endosomes
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To perform single-particle tracking analysis of Rab7-positive or Arl8b-positive endosomes, HeLaORF3a-Strep cells seeded on glass-bottom tissue culture-treated live-cell imaging dishes (ibidi) were either left untreated or Dox-treated (1 µg/mL) for 8 h in complete DMEM medium in a cell culture incubator. After 8 h, the cells were co-transfected with GFP-Rab7 and Arl8b-tomato expressing plasmids and incubated further for 16 h in complete DMEM with or without Dox (1 µg/mL). Before the start of the time-lapse confocal imaging, cells were washed with 1X PBS, phenol red-free DMEM media (Gibco) supplemented with 10% FBS was added, and live-cell imaging was performed using a ZEISS LSM 980 Elyra 7 super-resolution microscope with a 63×/1.4 NA oil immersion objective. To measure the mobile fraction and average speed of Rab7- or Arl8b-positive endosomes from time-lapse images, the “TrackMate” plugin of Fiji software was used with the following parameters:
•Vesicle diameter, 1 µm
•Detector, DoG
•Initial thresholding, none
•Tracker, Simple LAP tracker
•Linking max distance, 2 μm
•Gap-closing max distance, 2 μm
•Gap-closing max frame gap, 2
•Filters, none
After imaging, all the data were exported to a Microsoft Excel spreadsheet (2016) for further analysis.
•Vesicle diameter, 1 µm
•Detector, DoG
•Initial thresholding, none
•Tracker, Simple LAP tracker
•Linking max distance, 2 μm
•Gap-closing max distance, 2 μm
•Gap-closing max frame gap, 2
•Filters, none
After imaging, all the data were exported to a Microsoft Excel spreadsheet (2016) for further analysis.
4
HEK293-T Cell Transfection Optimized
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Human Embryonic Kidney (HEK293-T) cell were purchased from ATCC® (USA), and cultured in phenol red free DMEM media (Gibco, UK) supplemented with 10% FBS, with non-essential amino acids, 1% penicillin–streptomycin and 2 mM Glutamine. For live-cell imaging, cells were seeded in 8 well glass bottom chamber slides (Ibidi) at concentrations of 2.5 × 104 cells per well, 1 day before transfection. Cells were transfected with Fugene-HD transfection reagent (Promega, UK), as detailed by the manufacturer. 100–200 ng of DNA in 10 µl serum free, phenol red free DMEM media, mixed with 0.25 to 0.4 µl of transfection reagent and left in the tube with transfection complex for 10 min at room temperature. Then the transfection complex was added into wells containing cells, gently shaken and placed in a 37 °C incubator for at least 24 h.
5
Generating Stable Cell Lines with Biosensors
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Stable cell lines were generated using either HEK293 (ATCC, Manassas, VA) or HEK293–6E (National Research Council, Canada) cells20 (link). Briefly, cells were transiently transfected with 2CS, 1CS, or G32R null biosensor plasmids using Lipofecatime 3000 or 293fectin (Thermo Fisher Scientific). Flow cytometry was used to select and enrich for the population of cells expressing respective biosensors. Stable HEK293 cell lines were maintained in phenol red-free DMEM media (Gibco, Waltham, MA) supplemented with 2 mM GlutaMAX (Gibco, Waltham, MA), 10% fetal bovine serum (FBS) (Atlanta Biologicals, Lawrenceville, GA), 1 IU/mL penicillin/streptomycin (Gibco, Waltham, MA), and 250μg/mL G418 (Fisher Scientific). Stable HEK293–6E cell lines were maintained in F17 media (Sigma Aldrich) supplemented with Kolliphor p188 (Sigma Aldrich, St. Louis, MO), 200 nM/mL GlutaMAX, and either 1 μg/mL puromycin (Invitrogen, Carlsbad, CA) or 2μg/mL blasticidin (Goldbio) as a selection antibiotic. All cell lines were grown at 37°C with 5% CO2.
6
Lysosomal Membrane Permeability Assay
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The lysosomotropic stain, AO (Sigma-Aldrich), was used to determine LMP.26 , 27 (link) Cells were stained with AO (2.5 μg/ml; 12 min/37 °C), then washed twice with media and incubated with the copper complexes of Dp44mT (Cu[Dp44mT] 25 μM) or DpC (Cu[DpC] 10 μM) for 30 min/37 °C. Live cells were visualized in phenol-red-free DMEM media (Life Technologies) for green (495 nm excitation/516 nm emission) and red (577 nm excitation/592 nm emission) fluorescence using the Axio Observer.Z1 microscope and objective described above.
7
Measuring Cell Viability with AB1-42
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The viability of the cultures after treatment with Aβ1-42 was determined using the Cell-titre Blue assay (Promega, Southampton, UK). After experimental treatment, medium was removed from the wells of the 12-well cell-culture plate. Subsequently, the plate was washed with 500 μL phenol red-free DMEM media (Life Technologies), supplemented with 10% heat inactivated fetal bovine serum (NT2.N/A, NT2.A) or 10% horse serum (primary cortical cultures), 100 units/mL penicillin and 100 μg/mL streptomycin and 2 mmol/L L-glutamine. In all, 1 mL of Cell-titre blue reagent was mixed with 10 mL of the DMEM. In all, 500 μL of this solution was added to each well of the plate. The plate was incubated for 3 hours at 37°C. After incubation, medium was transferred to a 96-well plate and the absorbance was measured at 590 nm using a Thermo multiscan EX 96-well plate reader (Thermofisher, Loughborough, UK).
8
Breast Cancer Cell Line Antiproliferative Assay
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Antiproliferative activity evaluation of the synthesized compounds was assayed in two breast cancer cell lines. Thus, MCF-7 and MDA-MB-231 (American Type Tissue Culture Collection (ATTC), Rockville, MD, USA) were grown in Dulbecco′s Modified Eagle′s Medium (DMEM) media (Life Technologies, Gaithersburg, MD, USA), with 5% FBS (BioWest, Miami, FL, USA), 2 mM glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin. Cell cultures were incubated at 37 °C in a humidified atmosphere supplemented with 5% CO2 and 95% air. Prior to the treatment of the cells, MDA-MB231 and MCF-7 were grown in phenol red-free DMEM media (Life Technologies, Gaithersburg, MD, USA) containing 5% FBS. The FBS was charcoal-stripped (to eliminate the estrogenic effects) only for the MCF-7 cells.
9
Imaging cytoplasmic granules in live HeLa cells
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Live imaging was performed on HeLa cells grown on glass-bottomed dishes (MatTek Corporation) 48 h after cDNA transfection. The environment was kept at 37°C and 5% CO2. Cells were acclimated to the microscope incubator chamber before imaging. Only cells exhibiting low levels of TIA-1-GFP and devoid of spontaneous SGs were selected for subsequent SA treatment and imaging. Time-lapse movies of cells in DMEM phenol red–free media (#31053-028; Gibco) were taken using an inverted microscope (DMI 6000B; Leica) equipped with an HC Plan-Apochromat CS2 63×/1.40 NA oil objective lens and a 1394 ORCA-ERA camera (Hamamatsu Photonics) equipped with an incubation chamber and heated stage set to 37°C. Images were collected every 30 s for 10 min with maximum sample protection using two different channels using Volocity 6.1.1 software for acquisition. Analysis was made manually using the same software following individual PBs between frames. Only PBs and SGs that remained in focus for the 10 min of filming were included in the analysis.
10
Visualizing Lysosomes and Actin in U2OS Cells
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U2OS cells were seeded on coverglass in DMEM phenol red-free media (GIBCO BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a 5% CO2 incubator. Cell Light reagent (InvitrogenTM, Cat. No. C10507) was added and cells were cultured for 24 h. Then the cells were moved to microscopy, to imaged the transfected cells expressing the Lamp1-GFP-tagged lysosome. Next, the cells were washed twice with pre-warmed phosphate-buffered saline (PBS, pH 7.4) and fixed with 3.7% paraformaldehyde (PFA) in PBS for 10 min at room temperature. The fixed samples were washed twice with PBS. The supernatant was suctioned, the prepared dye (1 mL PBS: 25 μL Alexa Fluor 568 phalloidin of the 40× methanol stock solution, Cat. No. A12380) was added, and the cells were incubated for 1–2 h away from light. Finally, the samples were washed three times with PBS, and the plate was sealed.
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