Cf594

Manufactured by Biotium

CF594 is a fluorescent dye developed by Biotium. It has an absorption maximum at 593 nm and an emission maximum at 614 nm, making it suitable for detection in the orange-red region of the visible spectrum.

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Lab products found in correlation

Cf488, by Biotium (2 mentions) Docetaxel dtx, by Merck Group (1 mentions) Qdot 800, by Thermo Fisher Scientific (1 mentions) R1881, by Merck Group (1 mentions) Charcoal stripped fbs, by Thermo Fisher Scientific (1 mentions) Alexa conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions) Rpmi 1640, by Thermo Fisher Scientific (1 mentions) Penicillin, by Corning (1 mentions) Streptomycin, by Corning (1 mentions) Fetal bovine serum (fbs), by Corning (1 mentions) Rpmi 1640, by Corning (1 mentions) Lsm 510 laser scanning microscope, by Zeiss (1 mentions) Fetal bovine serum (fbs), by Cedarlane (1 mentions) Endothelial cell growth medium mv, by PromoCell (1 mentions) Orca charge coupled device, by Hamamatsu Photonics (1 mentions) Axiovert 200m microscope, by Zeiss (1 mentions) Tcs sp5, by Leica camera (1 mentions) Fitc conjugated anti mouse cd11b, by Thermo Fisher Scientific (1 mentions) Apc conjugated anti mouse tcrβ, by Thermo Fisher Scientific (1 mentions) Pe conjugated anti mouse cd8a, by Thermo Fisher Scientific (1 mentions)

7 protocols using cf594

Hormone-Sensitive LNCaP Cell Culture Protocol

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Hormone sensitive human PC cell line, LNCaP, cells were maintained in RPMI 1640 (Corning) supplemented with 10% fetal bovine serum (FBS) (Corning) and 100 IU/mL penicillin plus 100 mg/mL streptomycin (Corning) at 37°C with 5% CO₂. Before any experiments, cells were cultured in RPMI 1640 supplemented with 10% charcoal stripped FBS (Gibco) for 72 hours.
The following drugs were used for drug treatment: docetaxel (DTX; Sigma Aldrich), dihydrotestosterone (DHT) analog methyltrienolone, R1881 (Sigma Aldrich).
The following antibodies were used for immunofluorescence staining: mouse monoclonal anti-CD45 directly conjugated with QDot 800 (ThermoFisher Scientific), rabbit monoclonal anti-ARN21, rat monoclonal anti-tyrosinated-tubulin (Millipore Sigma), and mouse monoclonal anti pan-cytokeratin (BioLegend) directly conjugated with CF594 (Biotium). All Alexa-conjugated secondary antibodies were from Invitrogen.

Worroll D., Galletti G., Gjyrezi A., Nanus D.M., Tagawa S.T, & Giannakakou P. (2019). Androgen Receptor Nuclear Localization correlates with AR-V7 mRNA expression in Circulating Tumor Cells (CTCs) from Metastatic Castration Resistance Prostate Cancer Patients. Physical biology, 16(3), 036003.

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FISH Labeling of Chromosome TAD

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The bacterial artificial chromosomes (BACs) used for FISH labeling of a single TAD were a gift from E. Heard (19 (link)). They correspond to X chromosome “TAD H” characterized between genomic loci 102325818 and 102998976 [673,158 base pairs (bp)]. The four BACs required to cover this region are 6-RP24-217I10 (102302874:102459532), 7-RP23-469A2 (102465652:102651023), 8-RP23-331 L13 (102648562:102834423), and 9-RP24-396 M14 (102804354:102976347), spanning a total length of 673,473 bp. The 315-bp difference between BAC coverage and TAD boundary calling is considered negligible over the 673-kb total (<0.05% error). BAC probes were directly labeled by nick translation using CF-594 (Biotium).

Miron E., Oldenkamp R., Brown J.M., Pinto D.M., Xu C.S., Faria A.R., Shaban H.A., Rhodes J.D., Innocent C., de Ornellas S., Hess H.F., Buckle V, & Schermelleh L. (2020). Chromatin arranges in chains of mesoscale domains with nanoscale functional topography independent of cohesin. Science Advances, 6(39), eaba8811.

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Endothelial Cell Internalization of Extracellular Vesicles

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A semi-immortalized human bone marrow derived endothelial cell line (BMEC-1)^{62 (link)} was cultured in Endothelial Cell Growth Medium MV (Promocell) supplemented with 10% fetal bovine serum (Cedarlane, Burlington, Canada) and maintained at 37 °C under 5% CO₂.
EV internalization assays with BMEC-1. Purified EVs were labelled with PKH67 red fluorescent labelling kit (Sigma-Aldrich) and incubated with BMECs for 12 h before washing cells three times to remove unbound EVs. For uptake inhibition experiments endothelial cells were co-incubated with different compounds, as described in Fig. 5. Cells were fixed, permeabilized and stained for F-actin with CF594 (Biotium) and with the nuclear dye DAPI (Sigma-Aldrich). Leica TCS SP5. Epifluorescence was performed on a Axiovert 200 M microscope (Carl Zeiss. Germany) equipped with a camera (Orca charge-coupled device; Hamamatsu Photonics, Japan) using a 40 Plan-Neofluar (NA 1.3) oil immersion objective. Intensity analysis was performed with the Axiovision 4.6.3 software. Background subtraction was performed for each field. Confocal microscopy was performed on a Zeiss LSM 510 Laser Scanning Microscope using a 63 Plan-Neofluar water-immersion objective.

Babatunde K.A., Mbagwu S., Hernández-Castañeda M.A., Adapa S.R., Walch M., Filgueira L., Falquet L., Jiang R.H., Ghiran I, & Mantel P.Y. (2018). Malaria infected red blood cells release small regulatory RNAs through extracellular vesicles. Scientific Reports, 8, 884.

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Transferrin Internalization Assay

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After aPKC inhibitor treatment, explants were incubated with fluorescently conjugated human transferrin-594 (CF 594; 25 μg/mL; Biotium) for 40 min. The tissue was then washed extensively with cold 1X PBS and fixed in 4% paraformaldehyde for 2 h on ice.

Patel K., Nguyen J., Shaha S., Brightwell A., Duan W., Zubkowski A., Domingo I.K, & Riddell M. (2023). Loss of polarity regulators initiates gasdermin-E-mediated pyroptosis in syncytiotrophoblasts. Life Science Alliance, 6(10), e202301946.

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Stromal Vascular Fraction Isolation and Characterization

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The stromal vascular fraction (SVF) was isolated from epididymal fat pads via collagenase digestion and differential centrifugation as previously described (Orr et al. 2013). The following primary fluorophore‐conjugated antibodies, along with isotype controls, were used to characterize ATM and AT T‐cell (ATT) populations: APC‐conjugated anti‐mouse F4/80, FITC‐conjugated anti‐mouse CD11b, PE‐conjugated anti‐mouse CD11c, APC‐conjugated anti‐mouse TCRβ, Alexa 700‐conjugated anti‐mouse CD4, PE‐conjugated anti‐mouse CD8a (all from eBioscience, San Diego, CA), and CF594 (Biotium, Fremont, CA)‐conjugated anti‐mouse CD163 (clone E10B10, provided by Cytoguide Aps, Aarhus, DK). Immediately prior to analysis, DAPI or propidium iodide was added to permit live/dead cell discrimination. Separate aliquots of SVF cells were used to characterize ATM and ATT cell populations. Flow cytometry was performed on a LSRFortessa flow cytometer (BD Biosciences, San Jose, CA) at the Vanderbilt Flow Cytometry Core Shared Resource, and data were analyzed using Cytobank.

Orr J.S., Kennedy A.J., Hill A.A., Anderson‐Baucum E.K., Hubler M.J, & Hasty A.H. (2016). CC‐chemokine receptor 7 (CCR7) deficiency alters adipose tissue leukocyte populations in mice. Physiological Reports, 4(18), e12971.

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Immunostaining of Kidney Calbindin and β-Galactosidase

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Frozen sections of kidney were mounted on SuperFrost Plus glass slides and incubated with primary antibody (monoclonal antibodies against Calbindin D28K (diluted 1:200, abcam ab 11426) and anti-β-galactosidase (diluted 1:1000, abcam ab936)) overnight at 4°C. They were then incubated with the secondary antibodies (goat anti-rabbit IgG (diluted 1:1000, CF594, BIOTIUM) and goat anti-chicken IgY (diluted 1:1000, CF488, BIOTIUM) respectively, at room temperature for 3 to 4 hours.

Nabeshima Y., Washida M., Tamura M., Maeno A., Ohnishi M., Shiroishi T., Imura A., Razzaque M.S, & Nabeshima Y.I. (2014). Calpain 1 inhibitor BDA-410 ameliorates α-klotho-deficiency phenotypes resembling human aging-related syndromes. Scientific Reports, 4, 5847.

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Immunofluorescence Labeling Protocol

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The following antibodies were used: rabbit-αRab11 (715300. Invitrogen), mouse-αTfR (10R-CD71aHU. Fitzgerald, Acton, MA), mouse-αHA (MMS-101P. Covance, Princeton, NJ), chicken-αGAPDH (GW22763. Sigma-Aldrich, Saint Louis, MO), Alexa-labelled secondary antibodies (Invitrogen), IRdye-labelled secondary antibodies (Li-Cor, Lincoln, NE). The fluorophores used for protein conjugation were: SeTau-647 (SeTa Biomedicalos, Urbana, IL), CF-488 and CF-594 (Biotium, Hayward, CA). Alexa-594-labelled WGA was from Thermo Fischer (Grand Island, NY). Nocodazole was from Sigma-Aldrich.

Perez Bay A.E., Schreiner R., Benedicto I., Paz Marzolo M., Banfelder J., Weinstein A.M, & Rodriguez-Boulan E.J. (2016). The fast-recycling receptor Megalin defines the apical recycling pathway of epithelial cells. Nature Communications, 7, 11550.

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