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Fluorobrite medium

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FluoroBrite medium is a cell culture medium formulated to promote the growth and maintenance of fluorescent protein-expressing cells. It is designed to optimize the expression and stability of fluorescent proteins in cell culture applications.

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Lab products found in correlation

Axio observer z1, by Zeiss (6 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions) Lsm 880, by Zeiss (4 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions) Trypsin edta, by Thermo Fisher Scientific (3 mentions) Glutamax, by Thermo Fisher Scientific (3 mentions) Lsm 780 confocal microscope, by Zeiss (3 mentions) Sp5 confocal microscope, by Leica (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Axiovert s100, by Zeiss (2 mentions) Lsm 880 confocal microscope, by Zeiss (2 mentions) Ldh d c 485, by PicoQuant (1 mentions) Symphotime 64, by PicoQuant (1 mentions) P35g 0.170 14 c, by MatTek (1 mentions) A1 scanning confocal microscope, by Nikon (1 mentions) Micro incubator, by Okolab (1 mentions) Dic plan apochromat vc objective, by Nikon (1 mentions) Tcs sp5, by Leica (1 mentions) Fluorobrite medium, by R&D Systems (1 mentions) Tcs sp8, by Leica (1 mentions)

Spelling variants of this product

FluoroBrite (40 citations) FluoroBrite DMEM medium (31 citations) FluoroBrite Dulbecco’s modified Eagle’s medium (6 citations) FluoroBrite DMEM live-cell imaging medium (5 citations) FluoroBrite™ Dulbecco’s Modified Eagle’s Medium (DMEM; (3 citations) FluoroBrite Dulbecco’s Modified Eagle medium (DMEM) (2 citations) FluoroBrite Dulbecco’s modified Eagle’s cell culture medium (2 citations)

40 protocols using fluorobrite medium

1

Live Cell Imaging of Mitotic and Nuclear Dynamics

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For mitotic delay analysis, U2OS H2B-GFP cells were grown onto glass-bottom 8-well slides (IBIDI, 80827). After indicated treatments, medium was replaced by FluoroBrite medium (Gibco, A1896702) and live-cell imaging was performed on the Leica spectral confocal microscope TCS SP5. Images were taken every 6 min and 33 s for a total time of 24 h using the 63x glycerol objective.
For lysosome trafficking analysis, U2OS cells stably expressing LAMP1-RFP and H2B-GFP were grown onto glass-bottom 8-well slides (IBIDI, 80827). After indicated treatments, medium was replaced by FluoroBrite medium (Gibco, A1896702) and live-cell imaging was performed on Carl Zeiss LSM880 confocal microscope. Images were taken every 5 min for a total time of 24 h using the 63x glycerol objective.
For toroidal nucleus and nuclear lamina reformation assays, U2OS cells stably expressing H2B-GFP and mCherry-LMNA were grown onto bottom-glass 8 chambers slides (IBIDI, 80827). After addition of FluoroBrite with indicated treatments, mitotic cell division was analyzed on a Carl Zeiss LSM880 confocal microscope. For toroidal nucleus formation, images were taken every 93 s for 16 h using the 63x glycerol objective. For nuclear envelope reformation experiment, images were taken every 5 min for 16 h using the 63x glycerol objective.
Almacellas E., Pelletier J., Day C., Ambrosio S., Tauler A, & Mauvezin C. (2020). Lysosomal degradation ensures accurate chromosomal segregation to prevent chromosomal instability. Autophagy, 17(3), 796-813.
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2

HIV-1 nuclear complex formation in HeLa cells

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Hela cells transfected with siCNTRL and siSec24C siRNA were plated (5×104 cells/well) on poly-L-lysine treated 8-well chamber slides (LabTek). VSV-G pseudotyped HIVeGFP viruses labeled with INmNG were used to infect the cells at MOI 1. Prior to virus binding, cell nuclei were stained for 10 min with 2 μg/ml of Hoechst-33342. Infection was synchronized by binding virus to cells through spinoculation for 30 min at 1500 × g, 12 °C. The cells were washed twice, virus entry was synchronously initiated by adding pre-warmed Fluorobrite medium (GIBCO) supplemented with 10% FBS and followed by culture in a CO2 incubator. At 4 hrs post-infection, cells were examined under a Zeiss LSM880 laser scanning confocal microscopes using a 63x/1.4NA oil-immersion objective for nuclear HIV-1 complexes. Z-stack images spaced by 0.5 μm were acquired from 4 fields of view. The number of INmNG puncta in the nuclei was determined off-line using ICY software. After image acquisition, the cells were moved back into the cell culture incubator for additional 20 hours. At 24 hours post infection, the cells were fixed with 2% PFA for 7 min. The infected cells from 4 fields of view (~300–400 cells/field) were imaged using a 10x air objective. The fraction of eGFP expressing cells per field of view was determined by normalizing to the total number cells identified by Hoechst-33342 nuclear stain.
Rebensburg S.V., Wei G., Larue R.C., Lindenberger J., Francis A.C., Annamalai A.S., Morrison J., Shkriabai N., Huang S.W., KewalRamani V., Poeschla E.M., Melikyan G.B, & Kvaratskhelia M. (2021). Sec24C is an HIV-1 host dependency factor crucial for virus replication. Nature microbiology, 6(4), 435-444.
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3

Visualizing CCR6 Dynamics via Time-Lapse Microscopy

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For time-lapse microscopy, the Flp-InTRex293 cells that carried a single copy of wild-type or various mutant CCR6 with C-terminal GFP-tag were treated with CCL20 (50 nM) in FluoroBrite medium (Gibco) while recording by timelapse microscopy for 30 minutes (Online Supplementary Figure S4). The CCR6 expression was quantified using Image J (https://theolb.readthedocs.io/en/latest/imaging-/measuring-cell-fluorescence-using-imagej.html).
For flow cytometry analysis, the Flp-InTRex293 cells with a single copy of the wild-type or mutant CCR6 expression construct were treated with CCL20 (50 nM), and an aliquot (100 mL) was taken out at the indicated times for measuring surface CCR6.10 (link),19 (link)
Korona B., Korona D., Zhao W., Wotherspoon A.C, & Du M.Q. (2022). CCR6 activation links innate immune responses to mucosa-associated lymphoid tissue lymphoma development. Haematologica, 107(6), 1384-1396.
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4

Lysosome Dynamics Imaging in Cells

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The cells were grown on 35 mm glass bottom dishes (Mattek Corporation, P35G-0.170 14-C). 24 h after seeding, cells were incubated for 30min with 1μM Lyso Flipper53 (link) in fluorobrite medium (Gibco, 21083027) supplemented with 10% FCS (without FCS for HPDE) for 30min before imaging. For live imaging, cells were maintained at 37°C and 5% CO2 in a micro-incubator (Okolab, Pozzuoli NA, Italy). LLOMe was used at 1mM. For FLIM imaging, a 100× 1.45 NA oil DIC Plan-Apochromat VC objective (Nikon) with a Nikon A1 scanning confocal microscope was used. Excitation was performed using a pulsed laser at 485 nm (PicoQuant, LDH-D-C-485) at 20 MHz, and the emission signal was collected between 550 and 650 nm using a gated PMA hybrid detector and a TimeHarp 260 Nano Dual TSCPC unit. FLIM images were analyzed using the magic wand tool in SymPhoTime 64 software (PicoQuant) and corresponding pixels were fitted with a dual exponential reconvolution model whereby the lifetime τ1 was extracted. Data are expressed as means ± SD.
Gupta S., Yano J., Mercier V., Htwe H.H., Shin H.R., Rademaker G., Cakir Z., Ituarte T., Wen K.W., Kim G.E., Zoncu R., Roux A., Dawson D.W, & Perera R.M. (2021). Lysosomal retargeting of Myoferlin mitigates membrane stress to enable pancreatic cancer growth. Nature cell biology, 23(3), 232-242.
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5

Thrombin-Induced Endothelial Cell Responses

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HAECs were first starved in EBM2 or Fluorobrite medium (Gibco, A1896701) for at least two hours prior to thrombin treatment for 10 minutes. Thrombin was used at 0.25 U/mL for all experiments except for Fig. 4d, which was 0.5 U/mL for 7-10 minutes prior to assay. Cells were treated with lysophosphatidic acid for 30 minutes prior to assay. Y27632, latrunculin B, para-amino blebbistatin, or 2DG containing samples were starved in EBM2 or Fluorobrite for one hour prior to thrombin treatment.
Wu D., Harrison D.L., Szasz T., Yeh C.F., Shentu T.P., Meliton A., Huang R.T., Zhou Z., Mutlu G.M., Huang J, & Fang Y. (2021). Single-cell metabolic imaging reveals a SLC2A3-dependent glycolytic burst in motile endothelial cells. Nature metabolism, 3(5), 714-727.
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6

HIV-1 nuclear complex formation in HeLa cells

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Hela cells transfected with siCNTRL and siSec24C siRNA were plated (5×104 cells/well) on poly-L-lysine treated 8-well chamber slides (LabTek). VSV-G pseudotyped HIVeGFP viruses labeled with INmNG were used to infect the cells at MOI 1. Prior to virus binding, cell nuclei were stained for 10 min with 2 μg/ml of Hoechst-33342. Infection was synchronized by binding virus to cells through spinoculation for 30 min at 1500 × g, 12 °C. The cells were washed twice, virus entry was synchronously initiated by adding pre-warmed Fluorobrite medium (GIBCO) supplemented with 10% FBS and followed by culture in a CO2 incubator. At 4 hrs post-infection, cells were examined under a Zeiss LSM880 laser scanning confocal microscopes using a 63x/1.4NA oil-immersion objective for nuclear HIV-1 complexes. Z-stack images spaced by 0.5 μm were acquired from 4 fields of view. The number of INmNG puncta in the nuclei was determined off-line using ICY software. After image acquisition, the cells were moved back into the cell culture incubator for additional 20 hours. At 24 hours post infection, the cells were fixed with 2% PFA for 7 min. The infected cells from 4 fields of view (~300–400 cells/field) were imaged using a 10x air objective. The fraction of eGFP expressing cells per field of view was determined by normalizing to the total number cells identified by Hoechst-33342 nuclear stain.
Rebensburg S.V., Wei G., Larue R.C., Lindenberger J., Francis A.C., Annamalai A.S., Morrison J., Shkriabai N., Huang S.W., KewalRamani V., Poeschla E.M., Melikyan G.B, & Kvaratskhelia M. (2021). Sec24C is an HIV-1 host dependency factor crucial for virus replication. Nature microbiology, 6(4), 435-444.
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7

CXCR4 and β1AR Receptor Visualization

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HEK293AD and CHO cells were seeded on glass coverslips in six-well plates and transfected with CXCR4-ECFP or β1AR-ECFP; 24 h after transfection, cells were washed three times with PBS, and then PBS was replaced with 1% BSA in FluoroBrite medium (Gibco). After 1 h incubation at 37 °C and 5% CO2, cells were washed three times with PBS and were further incubated at 37 °C and 5% CO2 with 20 μg/mL human CXCR4 Alexa Fluor 488-conjugated antibody (FAB173G, R&D Systems) in FluoroBrite medium supplemented with 1% BSA; 1 h after incubation, cells were washed three times with PBS and incubated in FluoroBrite medium supplemented with 1% BSA for 3 × 10 min. After the last incubation, cells were imaged with a Leica TCS SP8 confocal laser-scanning microscope. CFP was excited using a 405-nm diode laser at 10% power and detected within the emission window of 430 nm to 490 nm. Alexa Fluor 488 was excited using the 492-nm laser line of the WLL at 10% power, and detected within the emission window of 500 nm to 650 nm. Image size was 512 × 512 pixels with 50-nm pixel size and 4.88-μs pixel dwell time.
Işbilir A., Möller J., Arimont M., Bobkov V., Perpiñá-Viciano C., Hoffmann C., Inoue A., Heukers R., de Graaf C., Smit M.J., Annibale P, & Lohse M.J. (2020). Advanced fluorescence microscopy reveals disruption of dynamic CXCR4 dimerization by subpocket-specific inverse agonists. Proceedings of the National Academy of Sciences of the United States of America, 117(46), 29144-29154.
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8

Visualization of EV Uptake in HeLa Cells

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HeLa cells were cultured in Dulbecco’s
Modified Eeagle’s Medium (DMEM, Thermo Fisher, Waltham, MA,
USA), high glucose, supplemented with 10% fetal bovine serum (FBS;
Thermo Fisher) and 1% penicillin/streptomycin (complete growth medium)
at 37 °C in a 5% CO2 atmosphere. HeLa cells were seeded
in Lab-Tek 8-well chambered cover glasses (Thermo Fisher, Waltham,
USA) or on self-prepared chambers pasted on glass slides with 2-component
glue. The glass slides were coated with 5 μg/cm2 poly-d-lysine (PDL; Advanced BioMatrix, Carlsbad, CA, USA) or 1 μg/cm2 fibronectin (Sigma-Aldrich, St. Louis, MO, USA). Cells were
cultured in complete growth medium for 48 h (80–90% confluency).
Cells were washed once 2 h before the experiment, and the medium was
changed to low-fluorescence FluoroBrite medium (Gibco, Carlsbad, CA,
USA) supplemented with 1% EV-depleted FBS to reduce autofluorescence
and starve the cells. eGFP-CD63 EVs were diluted 1:100 in PBS, added
to the cells, and incubated for 30–60 min at 37 °C in
5% CO2 atmosphere. To label the outer cell membrane, anti-CD59
monoclonal antibody conjugated with AlexaFluor 647 (Thermo Fisher,
Waltham, MA, USA) was added for 10 min and washed prior to imaging.
Puthukodan S., Hofmann M., Mairhofer M., Janout H., Schurr J., Hauser F., Naderer C., Preiner J., Winkler S., Sivun D, & Jacak J. (2023). Purification Analysis, Intracellular Tracking, and Colocalization of Extracellular Vesicles Using Atomic Force and 3D Single-Molecule Localization Microscopy. Analytical Chemistry, 95(14), 6061-6070.
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9

Plasma Membrane Staining in CL4 Cells

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CL4 cells were incubated with a 1.5x concentration of CellMask-DeepRed plasma membrane dye (Molecular Probes) in Fluorobrite Medium (Gibco) supplemented with 10% FBS and 1% L-glutamine for 10 min at 37° C. Cells were subsequently imaged in supplemented Fluoroborite Medium to minimize background fluorescence.
Gaeta I.M., Meenderink L.M., Postema M.M., Cencer C.S, & Tyska M.J. (2021). Direct visualization of epithelial microvilli biogenesis. Current biology : CB, 31(12), 2561-2575.e6.
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10

Rapamycin-Induced Translocation Assay

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The procedure has been described in detail previously (Hammond et al., 2014 (link)). In brief, HEK293 cells cultured in 35-mm glass-bottom dishes (In Vitro Scientific) were transiently transfected with 1 µg total DNA, which included the Lyn11-FRB::CFP recruiter, FKBP12 phosphatases (Hammond et al., 2012 (link)), PKCζ::GFP, and Par-6::iRFP as indicated. After 22–26 h, cells were imaged in Fluoro-Brite medium (Life Technologies) using a Nikon A1R confocal laser scanning microscope though a 100×, NA/1.45 plan apochromatic objective lens. Time-lapse imaging started 2 min before bath addition of 1 µM rapamycin. CFP::Lyn11-FRB images were used to generate binary masks to define PM. PM localization of each reporter was then calculated from the ratio of fluorescence within the PM to the whole cell and was expressed relative to the average before rapamycin addition (Hammond et al., 2014 (link)).
Dong W., Lu J., Zhang X., Wu Y., Lettieri K., Hammond G.R, & Hong Y. (2020). A polybasic domain in aPKC mediates Par6-dependent control of membrane targeting and kinase activity. The Journal of Cell Biology, 219(7), e201903031.
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