Cd40 pe

Manufactured by Beckman Coulter

Sourced in United States

CD40-PE is a fluorescent-labeled monoclonal antibody that binds to the CD40 antigen on the surface of human cells. It is used in flow cytometry applications to identify and enumerate CD40-positive cells.

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Anti-CD40-PE (4 citations)

5 protocols using cd40 pe

Phenotypic Analysis of Cell Suspensions

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Phenotypic analysis of cell suspensions was performed by flow cytometry. Cells were stained with a panel of seven 8-colour antibody combinations (Table 1). The following 6 monoclonal antibodies were used as a “backbone” in all combinations: CD38-PerCP5.5, CD10-APC, CD19-APC-H7, CD5-V450, CD45-V500 (BD Biosciences) and CD27-PeCy7 (Beckman Coulter). All FITC- or PE-labelled antibodies were specific of a particular combination, i.e. CD20-FITC, CD44-FITC, CD24-PE, CD40-PE, (Beckman Coulter); CD43-FITC, CD81-FITC, CD86-FITC, CD21-PE, CD22-PE, CD23-PE, CD268 (BAFF-R)-PE, IgD-PE, (BD Biosciences) IgM-FITC (Dako). Clone and isotype specificity of these antibodies are detailed in S1 Table; the antibodies were used at the dilution recommended by the manufacturers.
Samples containing 10⁶ cells in a volume of 100 μl were incubated with the combination of antibodies at the supplier recommended concentration during 20 minutes in the dark, at 4°C. After erythrocytes lysis with NH₄Cl at 4°C for 5 minutes, samples were washed with HBSS medium (Gibco).
Analysis was performed using 3-laser, 8-colour BD FACSCanto II flow cytometer (BD Biosciences) and FACSDiva software version 6 (BD Biosciences). At least 10⁴ cells were acquired in the CD19⁺ gate. BD CompBeads (BD Biosciences) were used for compensation settings. Cytometer performances were checked daily using CST beads (BD Biosciences).

Clavarino G., Delouche N., Vettier C., Laurin D., Pernollet M., Raskovalova T., Cesbron J.Y., Dumestre-Pérard C, & Jacob M.C. (2016). Novel Strategy for Phenotypic Characterization of Human B Lymphocytes from Precursors to Effector Cells by Flow Cytometry. PLoS ONE, 11(9), e0162209.

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Phenotyping Dendritic and Proliferating Cells

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For phenotypic analysis of dendritic cells: Fluorochrome conjugated antibodies CD14-PC5, CD80-FITC, HLADR-ECD, CD40-PE and CD86-PE conjugated were purchased from Beckman Coulter Inc (Carlsbad, CA, USA). The antibody against maturation marker CD83-conjugated to APC was purchased from Bio Legend (San Diego, CA, USA).
For phenotyping proliferating PBMCs, anti-CD56-PE, anti-CD4-PC5 anti-CD8-APC, anti- CD25-ECD and anti-CD3-PC7 antibodies were purchased from Beckman Coulter Inc (Carlsbad, CA, USA). Anti-FOXP3-PE was purchased from BioLegendInc (San Diego, CA, USA). Flow cytometry was performed as described previously [27 (link)]. All the samples were acquired using MoFlo XDP flow cytometer configured with three different lasers-blue (488 nm), violet (405 nm) red (640 nm) analyzed using Summit 5.2 software (both Beckman Coulter Inc, Carlsbad, CA, USA).

Dhandapani H., Jayakumar H., Seetharaman A., Singh S.S., Ganeshrajah S., Jagadish N., Suri A., Thangarajan R, & Ramanathan P. (2021). Dendritic cells matured with recombinant human sperm associated antigen 9 (rhSPAG9) induce CD4+, CD8+ T cells and activate NK cells: a potential candidate molecule for immunotherapy in cervical cancer. Cancer Cell International, 21, 473.

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Multiparametric Flow Cytometry Panel

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The following immunostaining Ab reagents were used for flow cytometry analysis: Mouse-anti-human CD83-PE, CD86-PE, OX40L-PE, CD40L-PE, CD3-FITC, CD4-APC, CD8-APC, CD19-PE, CD3-PE, HLA-DR-FITC (all from BD Biosciences), CD40-PE, CD56-PE (Beckman Coulter, Indianapolis IN U.S.A.), CCR7-FITC (R&D Systems), CD14-PE, CD1c-PE (Miltenyi Biotech, San Diego, CA) and the respective matched isotype controls (BD Biosciences). Prior to analysis for expression of CD40L, isolated CD4⁺ and CD8⁺ T cells were stimulated for 24 h with anti-CD3/CD28 activating Dynabeads (Gibco, Life Technologies). Purity was determined by the exclusive expression of either CD4 or CD8 on the CD3⁺ gated lymphocytes. Purity of blood-isolated DC was delineated using the following gating strategy: lineage (CD3, CD14, CD19, CD56)⁻ and CD1c⁺ HLA-DR⁺. Analysis was performed using the BD Biosciences LSR Fortessa Cell Analyzer and FlowJo version 7.6 software.

Zaccard C.R., Watkins S.C., Kalinski P., Fecek R.J., Yates A.L., Salter R.D., Ayyavoo V., Rinaldo C.R, & Mailliard R.B. (2014). CD40L induces functional tunneling nanotube networks exclusively in dendritic cells programmed by mediators of type-1 immunity. Journal of immunology (Baltimore, Md. : 1950), 194(3), 1047-1056.

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Differentiation and Activation of Human moDCs

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Peripheral blood
mononuclear cells were isolated
from buffy coats obtained from HLA-A2.1⁺ healthy volunteers
after written informed consent and in agreement with institutional
guidelines using Ficoll density gradient centrifugation (Lymphoprep,
ELITechGroup). CD14⁺ monocytes and naïve CD8⁺ T cells were isolated using CD14 microbeads and the human
Naive CD8⁺ T cell isolation kit (both Miltenyi Biotec),
respectively. Monocytes were differentiated into immature monocyte-derived
DCs (moDCs) in 6 days using RPMI 1640 containing 10% FCS, 2 mM l-glutamine, 0.5% antibiotic–antimycotic enriched with
interleukin-4 (IL-4, 300 U/mL), and GM-CSF (450 U/mL) (both Miltenyi
Biotec). Isolated cells were stored in liquid nitrogen in 10% DMSO
(CryoSure) and 90% FBS.
Human moDCs were thawed from liquid
nitrogen. In vitro DC activation (24 and 48 h) in
alginate cryogels containing NY-ESO-1 PLGA NPs was studied, as described
for BMDCs. Soluble NY-ESO-1 peptide and TLR ligands were added to
the controls in the same amounts as is present in NPs. Flow cytometric
staining was performed using Zombie Violet Fixable viability dye for
30 min at 4 °C, followed by cell-surface staining with the following
antibodies for 30 min at 4 °C: CD14-FITC (Miltenyi Biotec), CD80-PerCP-eFluor710
(PharMingen), CD86-PeCy7 (PharMingen), and CD40-PE (Beckman).

Weiden J., Schluck M., Ioannidis M., van Dinther E.A., Rezaeeyazdi M., Omar F., Steuten J., Voerman D., Tel J., Diken M., Bencherif S.A., Figdor C.G, & Verdoes M. (2021). Robust Antigen-Specific T Cell Activation within Injectable 3D Synthetic Nanovaccine Depots. ACS Biomaterials Science & Engineering, 7(12), 5622-5632.

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Phenotypic Analysis of Dendritic Cells

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For phenotypic analysis of dendritic cells: Fluorochrome conjugated antibodies CD14-PC5 (CD-cluster of differentiation PC5-PE-Phycoerythrin cy5-cyanine, CD80-FITC, HLADR-ECD, CD40-PE and CD86-PE were purchased from Beckman Coulter Inc (CA, USA). The antibody against maturation marker CD83conjugated to APC was purchased from Bio Legend (San Diego, CA, USA) For phenotyping proliferating PBMCs, anti-CD56-PE, anti-CD4-PC5 anti-CD8-APC, anti-CD25-ECD and anti-CD3-PC7 antibodies were purchased from Beckman Coulter Inc. Anti-FOXP3-PE was purchased from BioLegend Inc. Flow cytometry was performed as described previously [25] . All the samples were acquired using MoFlo XDP ow cytometer con gured with three different lasers -blue (488nm), violet (405nm) red (640nm) analyzed using Summit 5.2 software (both Beckman Coulter Inc).

Dhandapani H., Jayakumar H., Seetharaman A., Ganesarajah S., Sundersingh S., Jagadish N., Suri A., Rajkumar T., & Dhandapani H. (2020). Dendritic cells matured with recombinant human sperm associated antigen 9 (rhSPAG9) induce CD4+, CD8+ T cells and activate NK cells: A potential candidate molecule for immunotherapy in cervical cancer.

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