Prior to labeling with cell-permeable fluorescent dyes, the channels were washed once with phosphate-buffered saline to remove any serum and then filled with pre-warmed serum-free Fluorobrite DMEM (Invitrogen). A 5 mL syringe (BD Falcon) filled with 5 µm Calcein Green AM (Invitrogen) and 1 µm Hoechst 33342 (Invitrogen) in serum-free Fluorobrite DMEM was connected to the central channel inlet. Syringes filled with 1 µm Hoechst 33342 (Invitrogen) in serum-free Fluorobrite DMEM were connected to the flanking channel inlets. The syringes were run for 1 h at 1 mL h−1 using a syringe pump (Fusion 720; Chemyx). The microfluidic device was placed in a microscope stage incubator (Pathology Devices Inc.) while the cells were being labeled with the dyes.
Fluorobrite dmem
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Japan, Germany
FluoroBrite DMEM is a cell culture medium formulated for use in fluorescence-based applications. It is designed to reduce background fluorescence and provide optimal performance for live cell imaging and other fluorescence-based experiments.
Automatically generated - may contain errors
Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (42 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (19 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (16 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (14 mentions)
Hoechst 33342, by Thermo Fisher Scientific (11 mentions)
L glutamine, by Thermo Fisher Scientific (8 mentions)
Hoechst 33258, by Thermo Fisher Scientific (6 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (6 mentions)
Volocity software, by PerkinElmer (5 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (5 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (5 mentions)
Poly l lysine (pll), by Merck Group (5 mentions)
Opti mem, by Thermo Fisher Scientific (5 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Penicillin, by Thermo Fisher Scientific (4 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (4 mentions)
Trypsin edta, by Thermo Fisher Scientific (4 mentions)
Sp8 confocal microscope, by Leica (3 mentions)
Fluorobrite dmem, by Merck Group (3 mentions)
Imagem, by Hamamatsu Photonics (3 mentions)
381 protocols using fluorobrite dmem
1
Fluorescent Cell Labeling in Microfluidics
2
Polymer-based Cell Viability Assay
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Explants were treated with polymers, polymer-drug conjugates, or free DEX for 1 or 4 days, washed three times with 1x PBS and incubated with Calcein-AM and Hoechst 33342 in Fluorobrite DMEM (all from Thermo Fisher Scientific, Waltham, USA) for 30 min, washed three times with Fluorobrite DMEM and then kept in Fluorobrite DMEM. The articular surface of the explants was imaged on a Leica SP8 confocal microscope (Leica Microsystems GmBH, Wetzlar, GER) and viability calculated as the fraction of Calcein-AM- to Hoechst-positive cells in two regions per disk with an average of 50 cells per analyzed region.
3
Qdot-Based Receptor Tracking Protocol
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Qdot labeling was implemented via a two-step protocol. After the cells were allowed 24 h to achieve receptor expression, labeling was carried out by first incubating the cells with anti-HA-Fab-biotin at 0.2 µg/mL for 10 min at 37 °C. Following three washes with warm DMEM FluoroBrite™ (Thermo Fisher Scientific, Waltham, MA, USA), cells were then incubated with 0.05 nM SavQdot655 diluted in warm DMEM FluoroBrite™ supplemented with 1% BSA for 5 min at room temperature, washed three times with warm DMEM FluoroBrite™, and used immediately for time-lapse image series acquisition. Time-lapse image series were obtained on an inverted Nikon-Ti Eclipse microscope system equipped with the Yokogawa CSU-X1 spinning disk confocal scanner unit, a heated stage, a 60× oil-immersion Plan Apo 1.4 NA objective, and the Andor DU-897 electron-multiplying charged-coupled device (EMCCD) camera (Oxford Instruments Concord, Concord, MA, USA). Qdots were excited using a 405 nm solid-state diode laser (23 mW), and the Qdot emission was collected through the 641 nm (±75 nm) emission filter. CellMask™ Deep Red PM molecules were excited using the 647 nm laser line (150 mW), and the emission was collected using the 700 nm (±37 nm) emission filter. Single Qdot tracking was performed at a sampling rate of ~30 Hz (Δt = 0.032 s) for 2000 frames.
4
CRISPR-EChO Kinetics in U2OS Cells
5
Quantifying Collagen Type I Expression
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The staining for COL1A was carried out using the ‘scar-in-a-jar’ model developed by Chen et al. with some modifications [20 (link)]. The cultured fibroblasts were blocked with 3% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA) in FluoroBrite DMEM (GIBCO®, Life Technologies, Grand Island, New York, NY, USA) for 30 min at room temperature. Primary antibody (10AB, monoclonal mouse anti-human collagen type I, Santa Cruz Biotechnology, Dallas, TX, USA) was added to the cells and incubated at 37 °C in 5% CO2 for 90 min followed by one time washing with FluoroBrite DMEM. A 4% paraformaldehyde (Sigma Aldrich, Saint Louis, MO, USA) in PBS was added to the cells and incubated for 10 min at room temperature and washed two times with FluoroBrite DMEM. Secondary antibodies (AlexaFluor 488 goat anti-mouse IgG, Life Technologies, Eugene, Oregon, OR, USA) was added and incubated for 30 min followed by a one-time wash with FluoroBrite DMEM. The nucleus was stained for 10 min at room temperature with Hoechst (Cat. No. H3570, Life Technologies, Carlsbad, CA, USA) and washed two times with PBS, leaving the last wash in the wells for imaging.
6
Monitoring Gene Expression Dynamics
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For ON time course, on day 0, U2OS cells stably expressing CRISPR-EChO and sgRNA dissociated in TrypLE without phenol red (Life Technologies 12563-011) were seeded at 35% confluence in 96-well μ-plate (Ibidi 89626) in FluoroBrite™ DMEM (ThermoFisher A1896702) supplemented with 10% FBS and GlutaMAX (ThermoFisher 35050-061). On day 1, two hours before imaging, cells were treated with ProLong™ Live Antifade Reagent (ThermoFisher P36975). 100 μM abscisic acid was added to cells immediately after the Time 0 image is taken by adding a 3x concentrated stock to the imaging well. For OFF time course, the same protocol was maintained with the following changes. 100 μM abscisic acid was added on Day 0 at time of seeding. On day 1, immediately before imaging, cells were washed 3 times with DPBS (Life Technologies 14190-250) then cultured in complete FluoroBrite DMEM media.
7
Imaging Polycomb Bodies in Live Cells
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To image Polycomb bodies in live cells, PCGF2-HaloTag SCC1DEG cells were plated on a gelatinised 35 mm Petri dish, 14 mm Microwell 1.5 coverglass dishes (MatTek) at least 5 hours prior to imaging. Cells were labeled with 500 nm Halo-JF549 (Grimm et al., 2015 (link)) for 15 min at 37°C, followed by three washes, changing medium to Fluorobrite DMEM (Thermo Fisher Scientific) for imaging, which was supplemented as described for general ESC culture above. Cells were incubated for a further 30 min in supplemented Fluorobrite DMEM with 10 μg/mL Hoechst 33258 (Thermo Fisher Scientific) at 37°C and washed once more before imaging. Imaging was performed with an IX81 Olympus microscope connected to a Spinning Disk Confocal system (UltraView VoX PerkinElmer) using an EMCCD camera (ImagEM, Hamamatsu Photonics) in a 37°C heated, humidified, CO2-controlled chamber. Z stacks were acquired using a PlanApo 100x/1.4 N.A. oil-immersion objective heated to 37°C, using Volocity software (PerkinElmer). PCGF2-HaloTag-JF549 was imaged with a 561 nm laser at 1.25 s exposure at 15% laser power, SCC1-AID-GFP with a 488 nm laser at 1 s exposure at 40% laser power, while Hoechst was imaged with a 405 nm laser at 250 ms exposure at 20% laser power. Z stacks were acquired at 150 nm intervals. A total of at least 28 cells were imaged per condition in two biological replicates.
8
Visualizing Polycomb Bodies in Live Cells
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To image Polycomb bodies in live cells, HaloTag-SUZ12;PRC1CPM or RING1B-HaloTag;PRC1CPM cells were plated on gelatinised 35 mm Petri dish, 14 mm Microwell 1.5 coverglass dishes (MatTek, #P35G-1.5-14-C) at least 5 h before imaging. Prior to imaging, cells were labeled with 500 nm JF549 (Grimm et al., 2017 (link)) for 15 min at 37°C, followed by 3 washes, changing medium to Fluorobrite DMEM (Thermo Fisher Scientific) supplemented as described for general ESC culture above. Cells were incubated for a further 30 min in supplemented Fluorobrite DMEM with 10 μg/mL Hoechst 33258 (Thermo Fisher Scientific) at 37°C and washed once more before imaging. Cells were imaged on an IX81 Olympus microscope connected to a Spinning Disk Confocal system (UltraView VoX PerkinElmer) using an EMCCD camera (ImagEM, Hamamatsu Photonics) in a 37°C heated, humidified, CO2-controlled chamber. Z stacks were acquired using a 100x PlanApo NA 1.40 oil-immersion objective heated to 37°C, using Volocity software (PerkinElmer). HaloTag-JF549 was imaged with a 561 nm laser at 1.25 s exposure at 15% laser power, while Hoechst was imaged with a 405 nm laser at 250 ms exposure at 20% laser power. Z stacks were acquired at 150 nm intervals.
9
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).
10
Visualization of RFP/GFP-PTRS Reporter Localization
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The expression vectors for HA-tagged RFP/FLAG-tagged-GFP-PTRS reporters were transfected into 293 T cells using the Lipofectamine 2000 reagent. 293 T cells were then incubated in a CO2 culture chamber for 24 h. The medium was then replaced by FluoroBrite DMEM (Thermo Fisher Scientific) with FBS (10%). Images were visualized using a BZ-X710 microscope (KEYENCE), and the average fluorescence intensity was analyzed using ImageJ software. For co-localization analysis of RBPs, expression vectors for the FLAG-tagged-GFP-hPTRS reporter and mCherry-tagged-RBPs were co-transfected into 293 T cells using Lipofectamine 2000 reagent. After 24 h, the medium was replaced by FluoroBrite DMEM supplemented with FBS (10%), followed by the addition of Hoechst 33342 dye (5 μg/mL; Thermo Fisher Scientific). Images were visualized using the BZ-X710 microscope. The details of the materials are described in Supplementary Table 1 .
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