Cd69 fitc fn50

Manufactured by BD

CD69-FITC (FN50) is a fluorescently-labeled antibody that binds to the CD69 cell surface antigen. CD69 is a cell activation marker expressed on various immune cell types. This product is intended for research use only.

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4 protocols using cd69 fitc fn50

Comprehensive Immune Cell Profiling

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PBMC were immunophenotyped using the following antibodies: CD3-PerCP (UCHT1), CD4-APC (OKT4), CD18-PE (TS1/18), CD19-PE (HIB19), NKG2D-APC (1D11), CX3CR1-FITC (2A9-1, all Biolegend), CD3-Pacific Blue (UCHT1), CD8-FITC (HIT8a), CD16-PE-Cy5 (3G8), CD56-PE, -Alexa Fluor 647 or -PE-Cy7 (B159), CD69-FITC (FN50) (all BD Biosciences), CD62L-FITC (LT-TD180) (ImmunoTools), GCR-Alexa Fluor 488 (D8H2) (Cell Signaling Technology) and ADRB2 pure (S364, RayBiotech). The antibody against ADRB2 was coupled to R-phycoerythrin (RPE) in house using the ABSelect BSA Removal Kit to clean and concentrate the pure ADRB2 antibody followed by fluorochrome coupling with the Lightning-Link® R-Phycoerythrin Conjugation Kit according to manufacturer’s instructions (Innova Biosciences).
All stainings were performed as surface-stainings for 30 min. at 4°C, with exception of the intracellular GCR staining that was performed in whole blood according to a standard ICS protocol (Cell Signaling Technology). All sample acquisitions were performed on an Accuri^TM C6 (immune cell subsets and NK function) or a LSRFortessa^TM (NK receptor phenotyping) flow cytometer (BD Biosciences).

Bigler M.B., Egli S.B., Hysek C.M., Hoenger G., Schmied L., Baldin F.S., Marquardsen F.A., Recher M., Liechti M.E., Hess C, & Berger C.T. (2015). Stress-Induced In Vivo Recruitment of Human Cytotoxic Natural Killer Cells Favors Subsets with Distinct Receptor Profiles and Associates with Increased Epinephrine Levels. PLoS ONE, 10(12), e0145635.

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Comprehensive Immune Cell Profiling

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All cell surface antibodies and isotype controls were used at the recommended dilution of 0.25 μg antibody per million cells. Peripheral Blood Mono-nuclear Cells (PBMC) were stained with antibodies to phenotypic and functional markers for 15 minutes at room temperature in the dark, washed twice and fixed for 5 minutes at 4°C with Cytofix Buffer (BD Biosciences, San Jose, CA). The following surface antibodies (with clones shown in parentheses) and their appropriate isotype controls were obtained from BD Biosciences unless otherwise noted: CD69 FITC (FN50), CD107a PE (H4A3), CD56 PERCP Cy5.5 (B159), CD57 APC (NK-1), CD16 APC-H7 (3G8), CD3 BV510 (UCHT1), CD4 APC-H7 (RPA-T4), CD8 PE (RPA-T8), PD-1 FITC (MIH4), HLA-DR PERCP Cy5.5 (L243), CD38 APC (HIT2), Lineage FITC (cocktail), CD83 PE (HB15e), CD11c BV421 (B-ly6), BDCA-4 APC (REA380, Miltenyi Corporation, Auburn CA), and CD40 APC-H7 (5C3). Intra-cellular staining for IFN-gamma BV421 (B27), TNF-alpha FITC (MAB11), MIP-1 beta APC-H7 (D21-1351) or p24 FITC (Kc57, Beckman Coulter, Pasadena CA) was carried out in 1X Perm/Wash Buffer (BD Biosciences) as described by the manufacturer. A minimum of two hundred thousand events were collected on a BD LSR-II Flow Cytometer and samples were subsequently analyzed with FlowJo v9 software (Tree Star Incorporated, Ashland OR).

Tomescu C., Colon K., Smith P., Taylor M., Azzoni L., Metzger D.S, & Montaner L.J. (2020). Persons Who Inject Drugs (PWID) Retain Functional NK cells, Dendritic cell Stimulation, and Adaptive Immune Recall Responses Despite Prolonged Opioid Use.. Journal of leukocyte biology.

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Comprehensive T Cell Immunophenotyping

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LIVE/DEAD Fixable Aqua Stain (Invitrogen), excited by violet 405 nm laser and detected by 512 nm emission channel, was used to determine the percentage of dead cells. The T cell phenotype was determined by staining with mAbs: CD3-APC efluor780 (SK7, eBiosciences), CD4-BV786 (SK3, Biolegend), CD8-V500 (RPA-T8, BD Biosciences), and CD28-BV711 (CD28.2, BD Biosciences). Activation status was determined using mAbs: PD-1-PE-eFlour610 (J105, eBioscience), CD38-BUV737 (HB7, BD Biosciences), CD69-FITC (FN50, BD Biosciences), and CD25-BUV395 (2A3, BD Biosciences). Cytolytic profile was determined using mAbs: TNF-α-APC (6401.1111, BD Biosciences), Granzyme B-BV421 (GB11; BD Biosciences), and IFN-γ-PE-cy7 (B27, BD Biosciences). Monoclonal Ab CD14 BUV805 (M5E2, BD Biosciences, Franklin Lake, NJ) was used to exclude monocytes from analysis. Intracellular staining (ICS) was performed using the BD Cytofix/Cytoperm kit according to the manufacturer’s protocols. All antibody-stained cells were fixed in 1% formaldehyde (Sigma) prior to sample acquisition on a Symphony flow cytometer (BD Biosciences). Gates for flow cytometric acquisition and analyses were based on “fluorescence-minus-one” (FMO) controls and single stain compensation controls.

Crawford C.L., Dalecki A.G., Perez M.D., Schaaf K., Wolschendorf F, & Kutsch O. (2020). A copper-dependent compound restores ampicillin sensitivity in multidrug-resistant Staphylococcus aureus. Scientific Reports, 10, 8955.

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Multiparametric Phenotyping of Immune Cells

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The following flow cytometry antibodies were used: CD11c APC-Vio770 (MJ4-27G12, Miltenyi Biotec), CD14-PerCP (TUK4, Miltenyi Biotec), CD86-phycoerythrin (PE)-Cy7 (2331, BD Biosciences), CD80-PE (L307.4, BD Biosciences), HLA-ABC-VioBlue (REA230, Miltenyi Biotec), HLA-DR/DP/DQ-fluorescein isothiocyanate (FITC) (Tu39, BD Biosciences), CD3-PerCP (SK7, BD Biosciences), CD56-PE (B159, BD Biosciences), CD8-APC (RPA-T8, BD Biosciences) CD107a-FITC (H4A3, BD Biosciences), CD107b-FITC (H4B4, BD Biosciences), CD69-FITC (FN50, BD Biosciences), MICA/B-PE (6D4, BD Biosciences), ULBP2/5/6 (FAB1298p, R&D Systems), IFNγ-BV421 (4S.B3, BD Biosciences), mouse immunoglobulin G1 (IgG1) κ isotype control (PE/FITC/PerCP/PE-Cy7/APC; MOPC-21, BD Biosciences), mouse IgG2a κ isotype control (FITC/PE; G155-178, BD Biosciences), and REA Control-VioBlue (REA293, Miltenyi Biotec).

Jennings V.A., Scott G.B., Rose A.M., Scott K.J., Migneco G., Keller B., Reilly K., Donnelly O., Peach H., Dewar D., Harrington K.J., Pandha H., Samson A., Vile R.G., Melcher A.A, & Errington-Mais F. (2019). Potentiating Oncolytic Virus-Induced Immune-Mediated Tumor Cell Killing Using Histone Deacetylase Inhibition. Molecular Therapy, 27(6), 1139-1152.

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