Fluorobrite media, by Thermo Fisher Scientific - Product details

1

Quantifying Cellular Uptake of Liposomes and MPs

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BMDCs, Splenocytes, BMDCs or RAWs were analyzed with a SP5 two photon confocal microscope. 100k cells were allowed to attach to the bottom of a 96 well plate in their respective cell culture media. The next day, cells were washed with HBSS then either incubated with DiD containing liposomes for 15 mins, washed and incubated with MPs for 15 mins ord incubated with MPs for 15 mins, washed and then incubated with antibodies. Cells were washed and placed in fluorobrite media (Gibco) with 10% HIFBS +1:2000 dilution of Hoest then analyzed by microscopy using relevant wavelengths/filters. Wide field images were taken using 20X lens. Single images were taken using a 60x lens.
Quantification of images were performed in Fuji ImageJ. Wide field images of >100 cells were isolated based on nuclear stain. Gates were drawn on individual cells, FRs identified by cells with top 5% of RFU signal in FITC channel and liposome RFU calculated. Three images each from three separate wells were analyzed for a total of 9 images and >900 total cells.

Kong D, & DePamphilis M.L. (2001). Site-Specific DNA Binding of the Schizosaccharomyces pombe Origin Recognition Complex Is Determined by the Orc4 Subunit. Molecular and Cellular Biology, 21(23), 8095-8103.

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2

Apoptosis Assay for Cell Lines

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Cells were seeded into 96-well plates (Corning 3610 or 3596) in 100 μL of Fluorobrite media (Gibco, #A18967–01) containing 5% FBS minimally in triplicate. Because of baseline differences in proliferation between cell types, A673, CHLA10, and PSaRC 318 cells were seeded at a starting cell count of 5,000, 8,000, or 10,000 cells per well, respectively. Cell treatment conditions are described in the individual experiments. For apoptosis assays, IncuCyte Caspase 3/7 green reagent (Essen BioScience, catalog no: 4440) was added to a final dilution of 1:1,000. Phase contrast images of the cells in standard culture conditions were obtained at 3- to 6-hour intervals using an IncuCyte S3 or IncuCyte Zoom (Essen BioScience). Green fluorescence images were additionally captured for apoptosis assays. Experiments were repeated minimally in technical and biologic triplicates.

Maurer L.M., Daley J.D., Mukherjee E., Venier R.E., Julian C.M., Bailey N.G., Jacobs M.F., Kumar-Sinha C., Raphael H., Periyapatna N., Weiss K., Janeway K.A., Mody R., Lucas P.C., McAllister-Lucas L.M, & Bailey K.M. (2022). BRCA1-Associated RING Domain-1 (BARD1) Loss and GBP1 Expression Enhance Sensitivity to DNA Damage in Ewing Sarcoma. Cancer Research Communications, 2(4), 220-232.

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3

Stress Granule Formation in U2-OS Cells

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U2-OS cells producing GFP-G3BP1 or Halo-G3BP1 were seeded at 0.25×10⁶ cells in imaging dishes. One day after seeding, cells were chemically transfected with 2 μg plasmid DNA (1 μg of the ACTB-RNA tag fusion mixed with 1 μg of the transfection marker pNLS-TagBFP) using the TransIT transfection system following manufacturer recommendations (Mirus). On the next day, cells were stained with Halo dye and loaded with the Cbl-fluorophore probe as described above. Media added after bead loading contained 0.5 mM sodium arsenite and cells were incubated at 37 °C/5% CO₂ for 30-45 min to induce stress granules. Cells were rinsed once in PBS and Fluorobrite media (Gibco), supplemented with 0.5 mM sodium arsenite, was used for live cell imaging. For correlative imaging where cells were first imaged live, followed by fixation and FISH/immunofluorescence imaging of the same cells, the following modifications were made: gridded imaging dishes (MatTek) were coated with 1 μg/mL fibronectin (Sigma) for 4 hours, following one rinse with full media, plasmid ACTB-(A_T)4x was used and 3 μL of 50 μM Cbl-5xPEG-ATTO 590 was bead loaded.

Braselmann E., Wierzba A.J., Polaski J.T., Chromiński M., Holmes Z.E., Hung S.T., Batan D., Wheeler J.R., Parker R., Jimenez R., Gryko D., Batey R.T, & Palmer A.E. (2018). A multicolor riboswitch-based platform for imaging of RNA in live mammalian cells. Nature chemical biology, 14(10), 964-971.

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4

Stress Granule Formation in U2-OS Cells

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U2-OS cells producing GFP-G3BP1 or Halo-G3BP1 were seeded at 0.25×10⁶ cells in imaging dishes. One day after seeding, cells were chemically transfected with 2 μg plasmid DNA (1 μg of the ACTB-RNA tag fusion mixed with 1 μg of the transfection marker pNLS-TagBFP) using the TransIT transfection system following manufacturer recommendations (Mirus). On the next day, cells were stained with Halo dye and loaded with the Cbl-fluorophore probe as described above. Media added after bead loading contained 0.5 mM sodium arsenite and cells were incubated at 37 °C/5% CO₂ for 30-45 min to induce stress granules. Cells were rinsed once in PBS and Fluorobrite media (Gibco), supplemented with 0.5 mM sodium arsenite, was used for live cell imaging. For correlative imaging where cells were first imaged live, followed by fixation and FISH/immunofluorescence imaging of the same cells, the following modifications were made: gridded imaging dishes (MatTek) were coated with 1 μg/mL fibronectin (Sigma) for 4 hours, following one rinse with full media, plasmid ACTB-(A_T)4x was used and 3 μL of 50 μM Cbl-5xPEG-ATTO 590 was bead loaded.

Braselmann E., Wierzba A.J., Polaski J.T., Chromiński M., Holmes Z.E., Hung S.T., Batan D., Wheeler J.R., Parker R., Jimenez R., Gryko D., Batey R.T, & Palmer A.E. (2018). A multicolor riboswitch-based platform for imaging of RNA in live mammalian cells. Nature chemical biology, 14(10), 964-971.

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5

Fluorescence Imaging of Focal Adhesions

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Cells cultured on fibronectin-coated glass-bottom plates were washed with PBS and the DMEM culture media was replaced with warm FluoroBrite media (Gibco) supplemented with 10% FBS and 1:1,000 Nano-Glo luciferase assay substrate (Furimazine, Promega). First, the donor and acceptor emissions were imaged at 100× using an IX83 inverted microscope (Olympus) equipped with an iXon Ultra 888 EM-CCD camera (Andor), using a filter wheel with Semrock light filters FF01-460/60 (NanoLuc, donor) and FF01-536/40 (mNeonGreen, acceptor). Integration time was 45 s for both acquisitions. Next, cells were imaged for mNeonGreen using an LED lighting source and a standard FITC cube to obtain a regular fluorescent image of the FA.

Ramirez M.P., Anderson M.J., Kelly M.D., Sundby L.J., Hagerty A.R., Wenthe S.J., Odde D.J., Ervasti J.M, & Gordon W.R. (2022). Dystrophin missense mutations alter focal adhesion tension and mechanotransduction. Proceedings of the National Academy of Sciences of the United States of America, 119(25), e2205536119.

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6

Time-lapse Imaging of RPE1-hTERT Cells

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RPE1-hTERT cells were plated as single cells in 24-well glass-bottom dishes (Cellvis) at a density of 20,000 cells/well. Cells were grown in FluoroBrite media (#A18967-01; Gibco) with 10% FBS, 2 mM L-glutamine, 1× pen/strep at 37°C in a humidified enclosure with 5% CO₂ (Okolab) and stimulated with 20 ng/ml doxycycline where indicated. Images were collected starting 4 h after plating, and cells were imaged for 72 h with images collected every 10 min using a Nikon Ti Eclipse inverted microscope (20× 0.75 NA dry objective lens) with the Nikon Perfect Focus system. Images were captured using an Andor Zyla 4.2 sCMOS detector with 12-bit resolution. All filter sets were from Chroma, CFP-436/20 nm; 455 nm; 480/40 nm (excitation; beam splitter; emission filter), YFP-500/20 nm; 515 nm; 535/30 nm; and mCherry-560/40 nm; 585 nm; 630/75 nm. Images were collected with the Nikon NIS-Elements AR software.

Limas J.C., Littlejohn A.N., House A.M., Kedziora K.M., Mouery B.L., Ma B., Fleifel D., Walens A., Aleman M.M., Dominguez D, & Cook J.G. (2022). Quantitative profiling of adaptation to cyclin E overproduction. Life Science Alliance, 5(5), e202201378.

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7

Quantifying Cellular Uptake of Liposomes and MPs

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BMDCs, Splenocytes, BMDCs or RAWs were analyzed with a SP5 two photon confocal microscope. 100k cells were allowed to attach to the bottom of a 96 well plate in their respective cell culture media. The next day, cells were washed with HBSS then either incubated with DiD containing liposomes for 15 mins, washed and incubated with MPs for 15 mins ord incubated with MPs for 15 mins, washed and then incubated with antibodies. Cells were washed and placed in fluorobrite media (Gibco) with 10% HIFBS +1:2000 dilution of Hoest then analyzed by microscopy using relevant wavelengths/filters. Wide field images were taken using 20X lens. Single images were taken using a 60x lens.
Quantification of images were performed in Fuji ImageJ. Wide field images of >100 cells were isolated based on nuclear stain. Gates were drawn on individual cells, FRs identified by cells with top 5% of RFU signal in FITC channel and liposome RFU calculated. Three images each from three separate wells were analyzed for a total of 9 images and >900 total cells.

Deak P., Studnitzer B., Ung T., Steinhardt R., Swartz M, & Esser-Kahn A. (2022). Isolating and targeting a highly active, stochastic dendritic cell subpopulation for improved immune responses. Cell reports, 41(5), 111563.

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8

FRET Imaging of Focal Adhesions

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Cells cultured on fibronectin coated glass bottom plates were washed with PBS and the DMEM culture media was replaced with warm FluoroBrite™ media (Gibco) supplemented with 10% FBS and 1:1000 Nano-Glo® Luciferase Assay Substrate (Furimazine, Promega). First, the donor and acceptor emissions were imaged at 100x using an IX83 inverted microscope (Olympus) equipped with an iXon Ultra 888 EM-CCD camera (Andor), using a filter wheel with Semrock light filters FF01-460/60 (NanoLuc, acceptor) . Integration time was 45 s for both acquisitions. Then cells were imaged for mNeonGreen using a LED lighting source and a standard FITC cube, to obtain a regular fluorescent image of the FA.

Ramírez M.P., Anderson M.J., Sundby L.J., Hagerty A.R., Wenthe S., Ervasti J., & Gordon W.R. (2021). Dystrophin modulates focal adhesion tension and YAP-mediated mechanotransduction.

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9

Cell Viability Assay Using Presto Blue

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Cell viability required 10 μL of the Presto Blue cell metabolic assay (Presto Blue, Invitrogen, Frederick, MD, USA) along with 90 μL of 2% Fluorobrite Media supplemented with 2% fetal bovine serum (FBS), 1% l-glutamine (2mM), 1% antibiotics penicillin (5000 U/mL), and streptomycin (2500 U/mL) (all Gibco, Invitrogen, CA, USA). The phenol red media was added to each well of 96-well plates for viability calculation. Plates were incubated at 37 °C in an incubator for 60–90 minutes. Only metabolically live cells provide a color change from blue to purple. Fluorescence quantification was provided using a 96-well plate reader Spectra Max M5 (Molecular Devices, Sunnyvale, CA, USA) with λ_ex = 560 nm and λ_em = 600 nm via the bottom of the plate.13 , 14 The viability (%) is defined as:

Viability (%) = \frac{mean fluorescence of treated cells}{mean fluorescence of control cells} \times 100

Maqsood M., Qureshi R., Ikram M., Ahmad M.S., Jabeen B., Asi M.R., Khan J.A., Ali S, & Lilge L. (2018). In vitro anticancer activities of Withania coagulans against HeLa, MCF-7, RD, RG2, and INS-1 cancer cells and phytochemical analysis. Integrative Medicine Research, 7(2), 184-191.

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10

Quantifying Cellular Oxidative Stress

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The cellular ROS content was measured using a fluorescence‐based assay using the cell‐permeable CM‐H₂DCFDA dye (Invitrogen) according to the manufacturer's instructions. Briefly, cells were plated in black wall, clear bottom 96‐well plates at 15,000 cells/well in DMEM complete media, and cultured for 24 h. An oxidative stress inducer, tert‐butyl hydrogen peroxide (tBHP) (Sigma‐Aldrich) was used in some experiments to increase oxidative stress, as indicated. For the assay, cells were transferred to FluoroBrite media (Invitrogen) and exposed to 10 µM CM‐H₂DCFDA for 30 min at 37℃. After washing with PBS, fluorescence was measured using a CLARIOstar plate reader at Ex/Em of 492–495/517–527 nm. The fluorescence intensity was normalized to the total protein from the same wells measured by the Bradford method. The results presented for ROS measurements are representative of three independent experiments.

Jog R., Chen G., Wang J, & Leff T. (2021). Hormonal regulation of glycine decarboxylase and its relationship to oxidative stress. Physiological Reports, 9(15), e14991.

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Fluorobrite media

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17 protocols using fluorobrite media

Quantifying Cellular Uptake of Liposomes and MPs

Apoptosis Assay for Cell Lines

Stress Granule Formation in U2-OS Cells

Stress Granule Formation in U2-OS Cells

Fluorescence Imaging of Focal Adhesions

Time-lapse Imaging of RPE1-hTERT Cells

Quantifying Cellular Uptake of Liposomes and MPs

FRET Imaging of Focal Adhesions

Cell Viability Assay Using Presto Blue

Quantifying Cellular Oxidative Stress

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