Log In Sign up
The largest database of trusted experimental protocols

Fixable viability dye ef506

Manufactured by Thermo Fisher Scientific
Visit Supplier
Sourced in United States

Fixable Viability Dye eF506 is a fluorescent dye used to stain and identify dead cells in flow cytometry applications. The dye binds to proteins in dead cells, allowing for the discrimination between live and dead cells during analysis.

Automatically generated - may contain errors

Lab products found in correlation

Phorbol myristate acetate (pma), by Merck Group (5 mentions) Ionomycin, by Merck Group (3 mentions) Golgistop, by BD (2 mentions) Lsrfortessa, by BD (2 mentions) Ionomycin, by BD (1 mentions) Facsaria cell sorter, by BD (1 mentions) Lsrii flow cytometer, by BD (1 mentions) Anti gata3 bv421, by BioLegend (1 mentions) Anti cd127 pe cy7, by Thermo Fisher Scientific (1 mentions) Cytoflex flow cytometer, by Beckman Coulter (1 mentions) Fix perm, by BD (1 mentions) Fix perm buffer kit, by BD (1 mentions) Golgiplug, by BD (1 mentions) Ionomycin, by Solarbio (1 mentions) Intracellular staining perm wash buffer, by BioLegend (1 mentions) Brefeldin a solution, by BioLegend (1 mentions) Facscanto 2, by BD (1 mentions) Dxflex, by Beckman Coulter (1 mentions) Anti cd8, by BD (1 mentions) Anti cd3, by BD (1 mentions)

Spelling variants of this product

Fixable Viability Dye (414 citations) Viability dye (78 citations) EF506 (18 citations) EF506-viability dye (2 citations)

9 protocols using fixable viability dye ef506

1

Immunophenotyping of PsA Synovial Fluid

Check if the same lab product or an alternative is used in the 5 most similar protocols
Cell pellets from PsA SF samples were stained for surface markers with or without density gradient centrifugation in accordance with standard practice. Peripheral blood from patients with early PsA and healthy volunteers were also lysed for red blood cells and stained for surface markers in the same manner as described above. For intracellular staining, parts of the cell pellets were stimulated for 4 hours with 50 ng/ml of PMA (Sigma‐Aldrich), 500 ng/ml of ionomycin, and GolgiStop (BD Biosciences). Cells were then stained for surface markers and Fixable Viability Dye eF506 (eBioscience no. 65‐0866‐14) according to the manufacturer's instructions. Cells were then fixed with 2% paraformaldehyde in phosphate buffered saline (PBS) and permeabilized with 0.5% saponin buffer (0.5% bovine serum albumin, 0.05% NaN3 in PBS).
Sorted CD4+ and CD8+ T cells were cultured or cocultured ex vivo for 72 hours and restimulated to examine intracellular staining as described above. An LSRII flow cytometer (BD Biosciences) was used to analyze samples, and results were processed with FlowJo software (TreeStar). Cells were sorted with a FACSAria cell sorter (BD Biosciences), and the purity of the sorted cell populations was found to be ≥98%.
Xu X., Davelaar N., Mus A., Asmawidjaja P.S., Hazes J.M., Baeten D.L., Vis M., Bisoendial R.J., Prens E.P, & Lubberts E. (2020). Interleukin‐17A Is Produced by CD4+ but Not CD8+ T Cells in Synovial Fluid Following T Cell Receptor Activation and Regulates Different Inflammatory Mediators Compared to Tumor Necrosis Factor in a Model of Psoriatic Arthritis Synovitis. Arthritis & Rheumatology (Hoboken, N.j.), 72(8), 1303-1313.
+ Open protocol
+ Expand
2

Phenotypic Characterization of Dendritic Cells and ILC2s

Check if the same lab product or an alternative is used in the 5 most similar protocols
DCs were assessed using the antibodies of anti-CD11c-PC5.5; anti-CD80-FITC; anti-HLA-DR-APC-Cy7 and anti-CD86-PE-Cy7. PBMCs or ILC2s were stained with Fixable Viability Dye-eF506 and the following specific mAbs: Lineage cocktail (CD2, CD3, CD14, CD16, CD19, CD56, CD235a, FceR1)-FITC (all from eBioscience, San Diego, Calif, USA); anti-CRTH2-PE (BD Pharmingen, USA) and anti-CD127-PE-Cy7 (eBioscience, San Diego, Calif, USA). For intracellular cytokine assay, PBMCs or ILC2s were stimulated with phorbol myristate acetate (PMA), ionomycin, and brefeldin A (BFA) for 5 h, and then, the cells were fixed, permeabilized, and stained with anti-IL-13-BV421 (BioLegend, San Diego, CA, USA). For GATA3 levels detection, the cells were processed with the FoxP3/Transcription Factor Staining Buffer Set before staining with anti-GATA3-BV421(BioLegend, San Diego, CA, USA). After staining, samples were analyzed on a CytoFLEX Flow Cytometer (Beckman Coulter, Hercules, CA, USA).
Liu X.Q., Peng Y.Q., Huang L.X., Li C.G., Kuang P.P., Chen D.H., Wu Z.C., He B.X., Zhou Z.R, & Fu Q.L. (2023). Dendritic cells mediated by small extracellular vesicles derived from MSCs attenuated the ILC2 activity via PGE2 in patients with allergic rhinitis. Stem Cell Research & Therapy, 14, 180.
+ Open protocol
+ Expand
3

Intracellular Cytokine Staining and Recall Response Assay

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
Single-cell suspensions were stained with antibodies against surface molecules. For intracellular cytokine staining, cells were stimulated with PMA (50 ng/ml; Sigma-Aldrich, MO) and ionomycin (500 ng/ml; Sigma-Aldrich, MO) in the presence of brefeldin A (GolgiPlug, BD Biosciences) for 4 hours before staining with antibodies against surface proteins followed by fixation and permeabilization by using the eBioscience Fix/Perm or BD Fix/Perm buffer kit and staining with antibodies against intracellular antigens. For recall response experiment, cells were stimulated with OVA257–264 peptide (10 ng/ml) overnight and then added GolgiPlug for 2 hours. Cells were analyzed on an LSRFortessa (33 (link)) flow cytometer, and data were analyzed using FlowJo X. Dead cells were excluded on the basis of viability dye staining (Fixable Viability Dye eF506, eBioscience).
Liu X., Hao J., Wei P., Zhao X., Lan Q., Ni L., Chen Y., Bai X., Ni L, & Dong C. (2022). SMAD4, activated by the TCR-triggered MEK/ERK signaling pathway, critically regulates CD8+ T cell cytotoxic function. Science Advances, 8(30), eabo4577.
+ Open protocol
+ Expand
4

Flow Cytometry of Activated Immune Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols
Single cells were labeled with antibodies against surface molecules for 15 min at 4°C. Dead cells were labeled with viability staining (Fixable Viability Dye eF506, eBioscience). For intracellular cytokine detection, cells were stimulated with PMA (50 ng/mL, Sigma) and ionomycin (750 ng/mL, Solarbio) in the presence of Brefeldin A Solution (1000×, Biolegend) for 6 h. After stimulation, the cells were fixed with fixative (Servicebio) for 30 min, followed by 1× Intracellular Staining Perm Wash Buffer (Biolegend), then labeled with antibodies. Samples were analyzed using an FACS Canto II flow cytometer (BD Biosciences, USA) and DxFLEX (Beckman Coulter, China). Fluorochrome-conjugated antibodies are listed in Supplementary Table S2.
Li C., Zhang Z., Cai Q., Zhao Q., Wu H., Li J., Liu Y., Zhao X., Liu J., Ping Y., Shan J., Yang S, & Zhang Y. (2024). Peripheral CX3CR1+ T cells combined with PD-1 blockade therapy potentiates the anti-tumor efficacy for lung cancer. Oncoimmunology, 13(1), 2355684.
+ Open protocol
+ Expand
5

Phenotyping T-Cell Subsets in Mouse Blood

Check if the same lab product or an alternative is used in the 5 most similar protocols
The expression of markers on T cells in blood of five mice in each group was determined by flow cytometry after surface staining with a mouse T lymphocyte subset antibody cocktail, which included anti-CD3, anti-CD4, and anti-CD8 (BD Biosciences, 558431), conjugated with either PE-Cy7, PE, or APC in accordance with standard procedures. Isotype controls were included to ensure correct compensation and confirm antibody specificity. Cells were also stained with FIXABLE VIABILITY DYE EF506 (eBioscience, 65-0866-14) to avoid false positive results. Flow cytometry was performed on a BD FACSVerse flow cytometer and data were analyzed using Flow jo 10.0 software.
Xu Y., Li J., Lin Z., Liang W., Qin L., Ding J., Chen S, & Zhou L. (2022). Isorhamnetin Alleviates Airway Inflammation by Regulating the Nrf2/Keap1 Pathway in a Mouse Model of COPD. Frontiers in Pharmacology, 13, 860362.
+ Open protocol
+ Expand
6

Comprehensive Immunophenotyping of Single Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols
Single cells were suspended in PBS and incubated with antibodies against surface molecules for 30 min at 4°C. Dead cells were excluded based on viability dye staining (Fixable viability dye eF506; eBioscience). For intracellular cytokine detection, cells were stimulated with PMA (50 ng/ml; Sigma-Aldrich) and ionomycin (500 ng/ml; Sigma-Aldrich) in the presence of Brefeldin A (Golgiplug, BD Bioscience) for 5 h prior to staining. For intracellular staining, surface-stained cells were fixed and permeabilized with the Foxp3/Transcription Factor Staining Buffer Set (eBiosciences), followed by incubation with fluorochrome-conjugated antibodies.
For the detection of phosphorylated STAT3, cells were cultured in a fresh medium supplied with cytokines for 30 min, resuspended in cold PBS, and stained with antibodies against surface molecules. Then the cells were fixed with Phosflow Lyse/Fix buffer (BD Bioscience), followed by 90% methanol permeabilization and staining with antibodies against phosphorylated STAT3 in PBS. For analysis, the cells were acquired on an LSRFortessa (BD) flow cytometer, and data were analyzed using FlowJo 10.4.
Sun Q., Zhao X., Li R., Liu D., Pan B., Xie B., Chi X., Cai D., Wei P., Xu W., Wei K., Zhao Z., Fu Y., Ni L, & Dong C. (2023). STAT3 regulates CD8+ T cell differentiation and functions in cancer and acute infection. The Journal of Experimental Medicine, 220(4), e20220686.
+ Open protocol
+ Expand
7

Intracellular Cytokine Staining Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols
Single-cell suspensions were first stained with viability and surface marker antibodies. For intracellular staining of cytokines, both in vitro–cultured or in vivo–isolated cells were restimulated with phorbol 12-myristate 13-acetate (PMA; 50 ng/ml; Sigma-Aldrich) and ionomycin (500 ng/ml; Sigma-Aldrich) in the presence of GolgiStop (BD Biosciences) for 5 hours before surface staining and followed by fixation and permeabilization according to the manufacturer’s protocol. Cells were analyzed using LSRFortessa (BD Biosciences) flow cytometer and FlowJo X software. Dead cells were excluded on the basis of viability dye staining (Fixable Viability Dye eF506, eBioscience).
Chi X., Jin W., Zhao X., Xie T., Shao J., Bai X., Jiang Y., Wang X, & Dong C. (2022). RORγt expression in mature TH17 cells safeguards their lineage specification by inhibiting conversion to TH2 cells. Science Advances, 8(34), eabn7774.
+ Open protocol
+ Expand
8

Cytokine Profiling of T Cell Activation

Check if the same lab product or an alternative is used in the 5 most similar protocols
To measure intracellular levels, iT cells were cultured for 4 h at 1×106 cells/mL together with NALM6 at a 1:1 ratio in the presence of Brefeldin A (BD) monensin (BioLegend) and CD107a – BV421 (H4A3; BD). Cells were stained with ef506 Fixable Viability dye (ThermoFisher) prior to fixation and permeabilization using BD Cytofix/Cytoperm Plus kit as per manufacturer’s instructions, followed by staining with anti-cytokine and cell-surface antibodies GranzymeB – APC (GB12; Invitrogen), IFNγ – PE-Cy7 (4S.B3; Invitrogen), IL-2 – BUV737 (MQ1–17H12; BD), TNFα – PE (Mab11; Invitrogen), IL-17 – af488 (BL168, BioLegend), CD45 – BV605 (HI30; BioLegend). Percentage of cytokine producing cells was determined by flow cytometry.
To measure secreted cytokine levels, 0.5×106 T cells were cultured together with NALM6 at a 1:1 ratio or without target cells for 24 h. Supernatants were collected and stored at −80°C. Secreted cytokines were quantified using BD Cytometric Bead Array kits (IL-2 – 558270, IFNγ – 560111, TNFα – 560112) and flow cytometry.
van der Stegen S.J., Lindenbergh P.L., Petrovic R.M., Xie H., Diop M.P., Alexeeva V., Shi Y., Mansilla-Soto J., Hamieh M., Eyquem J., Cabriolu A., Wang X., Abujarour R., Lee T., Clarke R., Valamehr B., Themeli M., Riviere I, & Sadelain M. (2022). Generation of T-cell-receptor-negative CD8αβ-positive CAR T cells from T cell-derived induced pluripotent stem cells. Nature biomedical engineering, 6(11), 1284-1297.
+ Open protocol
+ Expand
9

Flow Cytometry Immunophenotyping Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols
The antibodies used for flow cytometry are described in Table S1. To discriminate between death and alive cells eF506 fixable viability dye (Thermo Fisher, Waltham, USA) was used. For the intracellular staining, cells were fixed and permeabilized using the Fixation/Permeabilization kit and Permeabilization buffer (ThermoFisher/ eBioscience). Samples were acquired on a FACSAria ™ III (BD Biosciences, Heidelberg, Germany) and data analyzed using FlowJo version 10 (Tree star Inc., Ashland, OR, USA).
Elizalde-Velázquez L.E., Schlosser-Brandenburg J., Laubschat A., Oser L., Kundik A., Adjah J., Groenhagen S., Kühl A.A., Rausch S, & Hartmann S. (2024). Th2-biased immune responses to body migrating Ascaris larvae in primary infection are associated with pathology but not protection. Scientific reports, 14(1).
+ Open protocol
+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!