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The TRFK5 is a laboratory instrument designed for time-resolved fluorescence (TRF) measurements. It provides a precise and sensitive method for quantitative analysis of fluorescent samples. The core function of the TRFK5 is to detect and measure the emission of fluorescent signals over time, enabling researchers to obtain accurate data for a variety of applications.

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Lab products found in correlation

Cytofix cytoperm fixation permeabilization solution, by BD (1 mentions) Brefeldin a, by Merck Group (1 mentions) Ionomycin, by Merck Group (1 mentions) Phorbol myristate acetate (pma), by Merck Group (1 mentions) Perm wash buffer, by BD (1 mentions) Absolute counting beads, by Thermo Fisher Scientific (1 mentions) Cd11b m1 70, by Thermo Fisher Scientific (1 mentions) Cd11c n418, by BioLegend (1 mentions) Cd3e 145 2c11, by BioLegend (1 mentions) Rm4 5, by Thermo Fisher Scientific (1 mentions) Klrg1 2f1, by Thermo Fisher Scientific (1 mentions) Novoexpress software, by Agilent Technologies (1 mentions) Rmst2 2, by Thermo Fisher Scientific (1 mentions) Celltrace violet, by Thermo Fisher Scientific (1 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions) Phosphate buffered saline (pbs), by Merck Group (1 mentions) Tc11 18h10, by BioLegend (1 mentions) Ebio13a, by Thermo Fisher Scientific (1 mentions) Xmg12, by BD (1 mentions) Rorγt q31 378, by BD (1 mentions)

4 protocols using trfk5

1

Cytokine Profiling of ILC2s

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To examine cytokine production in ILC2s, single-cell suspensions of SVF were cultured for 4h ex vivo with phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) (100 ng/ml) and ionomycin (Sigma-Aldrich) (1 μg/ml) in the presence of brefeldinA (10 μg/ml) (Sigma-Aldrich) in a 37°C incubator (5% CO2). Cells were washed with cold PBS and surface stained before fixation and permeabilization using the Cytofix/Cytoperm Fixation/Permeabilization Solution and Perm/Wash buffer according to the manufacturer’s protocol (BD Biosciences). Intracellular staining was performed with IL-5 (TRFK5) and IL-13 (eBIO13A) antibodies (eBioscience).
Mahlakõiv T., Flamar A.L., Johnston L.K., Moriyama S., Putzel G.G., Bryce P.J, & Artis D. (2019). Stromal Cells Maintain Immune Cell Homeostasis in Adipose Tissue via Production of Interleukin-33. Science immunology, 4(35), eaax0416.
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2

Flow Cytometric Characterization of ILC2s and Th2 Cells

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For the ILC2 analysis, the isolated MNCs were stained with a cocktail of the following antibodies: CD3e (145-2C11, BioLegend), CD11b (M1/70, eBioscience), CD11c (N418, BioLegend), CD45R (RA3-6B2, eBioscience), CD45 (30-F11, BioLegend), ST2 (RMST2-2, eBioscience), KLRG1 (2F1, eBioscience), and CD226 (10E5, BioLegend). ILC2s were characterized as a subpopulation of Lin (CD3e, CD11b, CD11c, and CD45R)− CD45+ cells that expressed KLRG1 and ST2. For intracellular cytokine staining, the MNCs were stimulated with PMA/Ionomycin/BFA for 5 h. After extracellular staining, the cells were fixed, permeabilized, and incubated with anti-IL-5 (TRFK5, eBioscience) and anti-IL-13 (eBioBA, eBioscience) antibodies. For Th2 cell analysis, CD4 (RM4-5, eBioscience), CCR6/CD196 (29-2 L17, BioLegend), and CCR4/CD194 (2G12, BioLegend) antibodies were used. The Th2 cells were defined as CD4+ CCR4+ CCR6− cells. Absolute counting beads (Invitrogen, USA) were employed to calculate the absolute cell number. FMO (Fluorescence Minus One) and matched isotype control were used as controls. The FCM data were obtained using a spectral cell analyzer (SONY SA3800) and analyzed with the Novoexpress software (Agilent Technologies).
Xie Y., Zhang Y., Zhu T., Ma J., Duan C., Yang L., Wang T., Zhuang R., Bian K, & Lu L. (2022). CD226 Deficiency Alleviates Murine Allergic Rhinitis by Suppressing Group 2 Innate Lymphoid Cell Responses. Mediators of Inflammation, 2022, 1756395.
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3

Allergen-Induced Pleural Inflammation

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Total BALBc or Il1rl1−/− PLEC were labelled with 5 μg ml−1 CFSE (Invitrogen) or 2 μM CellTrace Violet (Molecular probes), respectively, before injection of 200,000 labelled cells at a 1:1 ratio into the pleural cavity in 100 μl PBS. Fifty μg of Alt was delivered i.n. 18 h later. To block IL-5 within the pleural cavity, 30 μg of either purified functional grade anti-human/mouse IL-5 (eBioscience, clone TRFK5), 30 μg of Rat IgG1 (eBioscience, clone eBRG1) in 100 μl PBS (Sigma), or PBS (Sigma) alone was injected i.pl., immediately following injection 50 μl PBS or 50 μg of Alternaria extract (Greer) in 50 μl PBS (Sigma) was delivered i.n.
Jackson-Jones L.H., Duncan S.M., Magalhaes M.S., Campbell S.M., Maizels R.M., McSorley H.J., Allen J.E, & Bénézech C. (2016). Fat-associated lymphoid clusters control local IgM secretion during pleural infection and lung inflammation. Nature Communications, 7, 12651.
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4

Comprehensive Immune Profiling of Colon Tissue

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Single-cell suspensions were stained with the following antibodies: TCRβ (H57-597), NKp46 (29A1.4), NK1.1 (PK136) and fixable viability dye from eBioscience (San Diego, CA, USA); and CD4 (GK1.5), CD8 (53-6.7), CD19 (ID3), CD3ε (145-2C11), CD45 (30-F11), CD45.2 (104), CD90.2 (30-H12) and CD49a (Ha31/8) from BD Biosciences (Franklin Lakes, NJ, USA). Fixation and intracellular staining were performed using the Transcription Factor Staining Buffer Set (eBioscience) and antibodies against GATA-3 (TWAJ, eBioscience), RORγt (Q31-378, BD Biosciences) and EOMES (Dan11mag, eBioscience). Cytokine expression was determined by restimulation of cells isolated by digestion from the colon tissue in the presence of 100 ng/mL phorbol-12-myristate-13-acetate (PMA), 100 ng/mL ionomycin and 10μg/mL GolgiPlug™ and GolgiStop™ (BD Biosciences) in complete RPMI-1640 media (containing 10% heat-inactivated FCS, 1 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin and 50 µM β-mercaptoethanol) for 4 h. Cells were then fixed and stained for intracellular cytokines IFN-γ (XMG1.2, BD Pharmigen), TNF-α (Mab11, BD Pharmigen), IL-5 (TRFK5, eBioscience), IL-13 (eBio13A, eBioscience), IL-17A (TC11-18H10.1, BioLegend) and IL-22 (IL22JOP, eBioscience). Cells were analysed using a Fortessa X20 (BD Biosciences) and FlowJo software (Ashland, OR, USA) was used for the analysis.
Huang Q., Jacquelot N., Preaudet A., Hediyeh-zadeh S., Souza-Fonseca-Guimaraes F., McKenzie A.N., Hansbro P.M., Davis M.J., Mielke L.A., Putoczki T.L, & Belz G.T. (2021). Type 2 Innate Lymphoid Cells Protect against Colorectal Cancer Progression and Predict Improved Patient Survival. Cancers, 13(3), 559.
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