Cd16 fitc

Manufactured by Beckman Coulter

Sourced in United States

CD16-FITC is a fluorescently-labeled antibody that binds to the CD16 cell surface antigen. It is used to identify and quantify CD16-positive cells, such as natural killer cells and a subset of monocytes, in biological samples through flow cytometry analysis.

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CD16-FITC (3GB; CD3-FITC/(CD16+/CD56)-PE

7 protocols using cd16 fitc

Profiling T-cell activation and pSTAT5 signaling

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Flow cytometry and basal pStat5 experiments were performed according to previously published methods [17 (link), 41 (link)]. Monoclonal antibodies (mAbs) used were CD3-APCa750, CD4-ECD, CD8-APCa700, CD16-FITC, CD19-ECD, CD45RO-FITC, CD56-PCy7 and CD152-PE from Beckman Coulter; CD25-PE, CD45RA-APC and CD45RA-PCy7 from BD Biosciences; CD127-FITC from eBioscience; CD25-APC and LAP-PE from R&D Systems and GITR-PE from Miltenyi.
For pSTAT5 activation experiments, peripheral blood mononuclear cells (PBMCs) were cultured (500,000/well) for 30 min at 37° C and IL-2 was added for 15 min. Cells were then fixed with 1.6% paraformaldehyde, permeabilized with 100% methanol, stained for pSTAT5 and appropriate surface markers. Events were collected using a LSRFortessa flow cytometer (BD Biosciences) and data were analyzed using Diva software (BD Biosciences). To calculate EC50, after subtracting the value from the media control, binding data for each sample was normalized to 100% based on the maximal response and non-linear regression of these data was performed assuming a variable slope using Graph-Pad Prism 6.0.

Rosenzwajg M., Churlaud G., Mallone R., Six A., Derian N., Chaara W., Lorenzon R., Long A., Buckner J., Afonso G., Pham H.P., Hartemann A., Yu A., Pugliese A., Malek T.R, & Klatzmann D. (2015). Low-dose interleukin-2 fosters a dose-dependent regulatory T cell tuned milieu in T1D patients. Journal of autoimmunity, 58, 48-58.

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Plasmacytoid Dendritic Cell Enumeration in PBMCs

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The percentage of pDCs in PBMCs was obtained
by a three-color flow cytometric method
within 4 hours of PBMC preparation. A mixture of
monoclonal antibodies specifically established to
identify pDCs was used. Cells were stained with
CD14-FITC, CD16-FITC, CD85k (ILT3)-PE,
and CD123-PC5 according to the manufacturer’s
instructions (Beckman Coulter, Brea, CA, USA).
pDCs were identified as being CD14lo/-CD16lo/-
CD85k (ILT3)± and CD123± (10 (link)). Briefly, 100 μL
of PBMCs were incubated with a mixture of the
monoclonal antibodies (20 μL) for 20 minutes
at room temperature (RT) in the dark. After two
washes with PBS, stained aliquots of PBMCs were
reconstituted in 0.5 mL of PBS. Flow cytometry
readings of the stained cells were acquired using
Cytomics FC 500 (Beckman Coulter, Brea, CA,
USA) with CXP Software (Fig 1).
Absolute pDC numbers were estimated by multiplying
the percentage of pDCs by the number of
PBMCs, which was calculated by multiplying the
number of white blood cells (WBCs) by the percentage
of lymphocytes plus monocytes in WBCs
as measured in the Sysmex XE-2100 apparatus
(Sysmex Corporation; Kobe, Japan).

Cho C.H., Yoon S.Y., Lee C.K., Lim C.S, & Cho Y. (2015). Effect of Interleukin-29 on Interferon-α Secretion by Peripheral Blood Mononuclear Cells. Cell Journal (Yakhteh), 16(4), 528-537.

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Quantifying Immune Cell Subsets

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The PBMC were incubated with combinations of fluorescein isothiocyanate (FITC), phycoerythrin (PE), PE-cyanine 5.5 (PE-cy5.5), and peridinin chlorophyll protein monoclonal antibodies. The monoclonal antibodies were CD3-FITC, CD3-Percp/Cy5.5, CD4-PE, CD4-FITC, CD8-FITC, CD8-PE, CD16-FITC, CD56-PE, CD19-PE, CD25-FITC, CD127-Percp/Cy5.5, and CD28-PE (Beckman Coulter). About 10,000 lymphocytes were assessed with FC500 software to determine the percentage of CD3⁺, CD4⁺, CD8⁺, CD3⁻CD16⁺CD56⁺, CD19⁺, CD4⁺CD25⁺CD127⁺, CD8⁺CD28⁻, and CD8⁺CD28⁺ lymphocytes.

Yao J., Guan S., Liu Z., Li X, & Zhou Q. (2020). Changes in immune indicators and bacteriologic profile were associated with patients with ventilator-associated pneumonia. Medicine, 99(16), e19716.

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Comprehensive Immune Cell Phenotyping

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The following antibodies were used in this study: CD3 AF700, CD4 BV421, CD4 FITC, CD6 APC, CD10 BV605, CD19 APC-Cy7, CD21 PE-Cy7, CD25 PerCP-Cy5.5, CD27 BV421, CD28 PerCP-Cy5.5, CD38 PerCp-Cy5.5, CD45RA APC-Cy7, TCR αβ PerCP-Cy5.5, IL-2 PE, LAT PE, NTAL/LAB APC, and biotin anti–human TCR-Vd2 (BioLegend); TCR-Vd1 APC (Miltenyi Biotec); CD8 APC, CD8 Pacific blue, CD21 PE-Cy7, CD31 PE, CD56 APC, CD69 FITC, CD107a PE, CD127 Alexa Fluor 647, IFN-γ FITC, TCR γδ PE, IgG Alexa Fluor 700, IκBα PE, ERK1/2(pT202/pY204) AF647, and ZAP70(pY319)/SYK(pY352) APC (BD); IgD FITC and IgA PE (SouthernBiotech); CD3 PE-Cy7, CD4 PE-Cy7, CD8 PE, CD16 FITC, CD45RA FITC, CD45 Pacific blue, Vα24 PE, and Vβ11 FITC (Beckman Coulter); CCR7 PE (R&D Systems); Bruton tyrosine kinase/ITK(pY551/pY511) PE, IL-17 PE, IL-4 APC, and ICOS PE (eBioscience); IgM Alexa Fluor 647 (Jackson ImmunoResearch Laboratories, Inc.); and PLCγ1(pY783) and goat anti–rabbit AF647 (Cell Signaling Technology). For immunohistochemistry, CD21 (Dako), CD20 (Invitrogen), CD3 (Cell Marque), CD4 and CD8 (Spring Bioscience), and Bcl-6 (Leica Biosystems) were used. For immunoblotting LAT (sc-7948; Santa Cruz Biotechnology, Inc.), FLAG tag (AHP1074; AbD Serotec), actin (sc-1616; Santa Cruz Biotechnology, Inc.), PLCγ1 (pY783; no. 2821; Cell Signaling Technology), and ZAP70 (pY319; no. 2701; Cell Signaling Technology) were used.

Keller B., Zaidman I., Yousefi O.S., Hershkovitz D., Stein J., Unger S., Schachtrup K., Sigvardsson M., Kuperman A.A., Shaag A., Schamel W.W., Elpeleg O., Warnatz K, & Stepensky P. (2016). Early onset combined immunodeficiency and autoimmunity in patients with loss-of-function mutation in LAT. The Journal of Experimental Medicine, 213(7), 1185-1199.

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Multiparametric Flow Cytometry Analysis

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Cells were phenotypically analyzed using a Navios™ instrument with 10-color PMTs and three solid-state lasers (Beckman Coulter, Fullerton, FL). The list mode data files were further analyzed using Kaluza™ software (Beckman Coulter). In order to guarantee that the optics, laser, fluidics and fluorescence intensity were stable during all measurements calibration was performed using Flow Check Pro Fluorospheres (Beckman Coulter) and Cyto-Cal Multifluor + Violet Fluorescence Alignment Beads (Thermo Scientific, Fremont, CA). Cells were washed with PBS with 1% bovine serum albumin before being labelled with fluorochrome-conjugated mAbs. After incubation for 30 min at 4°C in the dark, cells were washed twice to remove unbound antibodies and analyzed. For cell surface staining, the following mAbs were used: CD3-ECD (A07748), CD16-FITC (IM0814U), CD45-Krome Orange (A96416), CD56-APC-Alexa Fluor750 (custom made), CD158a-APC-Alexa Fluor700 (custom made), CD158b-PC7 (A66901), CD158e1/e2-APC (A60795), CD159a-PC5.5 (custom made) (all from Beckman Coulter, Marseille, France) and CD159c-PE (FAB138P; R&D).

Kleinnijenhuis J., Quintin J., Preijers F., Joosten L.A., Jacobs C., Xavier R.J., van der Meer J.W., van Crevel R, & Netea M.G. (2014). BCG-induced trained immunity in NK cells: role for non-specific protection to infection. Clinical immunology (Orlando, Fla.), 155(2), 213-219.

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Multiparameter flow cytometric analysis

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For the analysis of OX40L expression, whole blood samples were stained with anti-CD14-PC5, CD16-FITC, CD11c-APC, HLA-DR-PC7, and OX40L-PE mAbs, and red blood cells were lysed with Versalyse (Beckman Coulter). For the analysis of blood Tfh cells, whole blood samples were stained with anti-CXCR5-AF488, CCR6-PE, CXCR3-PC5, CCR4-PC7, CD3-AF700, CD8-APCH7, CD4-Pacific Blue (all from Becton Dickinson), CD45RA-ECD (Beckman Coulter), ICOS-APC (Biolegend) and CD45-Pacific Orange (Invitrogen). Data were collected using a BD LSR II instrument (BD Biosciences) and analyzed with Flowjo software (Tree Star Inc.).

Jacquemin C., Schmitt N., Contin-Bordes C., Liu Y., Narayanan P., Seneschal J., Maurouard T., Dougall D., Davizon E.S., Dumortier H., Douchet I., Raffray L., Richez C., Lazaro E., Duffau P., Truchetet M.E., Khoryati L., Mercié P., Couzi L., Merville P., Schaeverbeke T., Viallard J.F., Pellegrin J.L., Moreau J.F., Muller S., Zurawski S., Coffman R.L., Pascual V., Ueno H, & Blanco P. (2015). OX40 Ligand Contributes to Human Lupus Pathogenesis by Promoting T follicular Helper Response. Immunity, 42(6), 1159-1170.

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Immunophenotyping of Leukocyte Subsets

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The flow cytometric analysis (FCM) of leukocyte populations were performed by three-color fluorescence methodology. Peripheral blood cells were stained with antibodies to CD45/PerCP (BD Biosciences, CA, USA), CD3/PE (BD), CD4/FITC (BD), CD8/PE (BD), CD16/FITC (Beckman Coulter, CA, USA), CD56/PE (BD), CD19/FITC (BD), CD20/PE (BD), and CD22/PE (Dako, Tokyo, Japan). After gating lymphocytes by CD45/side-scatter, expression profiling of CD markers was performed using a FACS Canto II (BD) and was analyzed using the BD FACS Diva software program (ver.6.1.2; BD).

Fukui S., Hidaka M., Fukui S., Morimoto S., Hara T., Soyama A., Adachi T., Matsushima H., Tanaka T., Fuchigami M., Hasegawa H., Yanagihara K, & Eguchi S. (2021). The Contribution of Serum Complement Component 3 Levels to 90-Day Mortality in Living Donor Liver Transplantation. Frontiers in Immunology, 12, 652677.

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