Cd40l pe

Manufactured by BD

Sourced in United States

CD40L-PE is a lab equipment product that is used for the detection and analysis of CD40L, a protein involved in various cellular processes. It is a fluorescently labeled antibody that binds to CD40L, allowing for its identification and quantification in samples.

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CD40L (12 citations) PE-conjugated anti-CD40L mAb (2 citations) Anti CD40L-PE (2 citations)

6 protocols using cd40l pe

Investigating Immune Checkpoint Modulation in PBMCs

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AIM assays were performed as previously described ^{43 (link)}. Briefly, cryopreserved PBMC were thawed, washed, resuspended in R10, and rested for 3 hours at 37°C. Following the 3-hour rest interval, the appropriate number of cells were transferred to a 48-well plate and subsequently treated with CD40 blocking antibody (Miltenyi Biotec, cat. no. 130–094-133) for a final concentration of 0.5ug/mL for 15 minutes at 37°C. Cells were incubated in the presence or absence of our 10-LRA panel, as previously described. After an 18hr incubation, cells were harvested and stained for 50 minutes at 4°C with the surface staining monoclonal antibodies (mAb); (PD-L1-PE/Cy7 (Biolegend, cat. no. 329717), CD40L-PE (BD Biosciences [BD], cat. no. 561720), OX40-APC (BD cat. no. 563473), CD69-BV650 (Biolegend cat. no. 310933), CD3-BV605 (Biolegend cat. no. 317322), CD4-BV421 (BD cat. no. 562424), CD8-PerCp-Cy5.5 (BD cat. no. 560662), CD25- BUV395 (BD cat. no. 564034) and LIVE/DEAD Near-IR stain (ThermoFisher cat. no. L34975), washed, fixed and permeabilized (BD cytofix fixation buffer, cat. no. 554655), and then stained for 30 minutes at 4°C with intracellular mAb Acetyl-histone H3-Alexa Fluor 488 (Cell Signaling Technology, cat. no. 9683S) in 1x perm/wash buffer (BD, cat. no. 554723). The cells were washed and fixed (BD cytofix fixation buffer, cat. no. 554655) prior to flow cytometry analysis.

Lilie T., Bouzy J., Asundi A., Taylor J., Roche S., Olson A., Coxen K., Corry H., Jordan H., Clayton K., Lin N, & Tsibris A. (2023). Opioid use does not limit potent low-dose HIV-1 latency reversal agent boosting. medRxiv.

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Multiparameter flow cytometry for cTfh subsets

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PBMCs were stained with CD4-FITC, CXCR5-PE, CXCR3-APC, CCR6-PE, CD27-FITC, CD38-PE, CD40L-PE, and IFNγ-PE antibodies (BDPharmingenTM, USA) and incubated at 4°C for 30 min in the dark. After the antigen–antibody incubation was completed, the specimen was cleaned 3 times with 2.5 mL of flowing washing solution (Hyclone, USA)/PBS, then resuspended in 500 μL of PBS, and put in storage at 4°C for performing upper flow cytometry analysis. The isotype control antibody was used to adjust the compensation of each channel and set the gate parameters. FlowJo software (Version 7.6.1, Tree Star Inc., USA) was employed to analyze the data obtained by flow cytometry. CD4+CXCR5+CXCR3+CCR6 indicates Th1-like cTfh cells, CD4+CXCR5+CXCR3-CCR6 indicates Th2-like cTfh cells, and CD4+CXCR5+CXCR3-CCR6+ indicates Th17-like cTfh cells.

Zhao G., Liang J., Cao J., Jiang S., Lu J, & Jiang B. (2022). Abnormal Function of Circulating Follicular Helper T Cells Leads to Different Manifestations of B Cell Maturation and Differentiation in Patients with Osteosarcoma. Journal of Healthcare Engineering, 2022, 3724033.

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Multicolor Flow Cytometry Analysis

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The fluorochrome-labeled monoclonal antibodies used in this study included: antibodies against CD3-Peridinin Chlorophyll (Percp, Miltenyi Biotec), CD8-APCcy (Biolegend), CD4-phycoerytherin (PE, BD Pharmingen), CD27-APCcy7 (BD Pharmingen), IgD-PEcy7 (Biolegend, San Diego, CA), CD40L-PE (BD Pharmingen), CD19- fluorescein isothiocyanate (FITC, BD Pharmingen), CD80-allophycocyanin (APC, BD Pharmingen), PD1-PE (BD Pharmingen), CD45RO-PEcy7 (BD Pharmingen), ki67-FITC (BD Pharmingen), IFN-γ-FITC (BD Pharmingen), CD38-APC, annexin V-FITC, and isotype control Abs (BD Pharmingen). No annexin V staining was used as a control for gating strategy. All others were gated based on isotypes. Cells were identified by their forward (FSC) and side scatter (SSC) characteristics and were analyzed with a Guava 8HT flow cytometer (Millipore, Billerica, MA).

Powell A.M., Luo Z., Martin L., Wan Z., Ma L., Liao G., Song Y., Li X., Kilby J.M., Huang L, & Jiang W. (2016). The induction of CD80 and apoptosis on B cells and CD40L in CD4+ T cells in response to seasonal influenza vaccination distinguishes responders versus non-responders in healthy controls and aviremic ART-treated HIV-infected individuals. Vaccine, 35(5), 831-841.

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Multiparameter Flow Cytometric Phenotyping

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Surface markers were evaluated using combinations of fluorochrome-conjugated monoclonal antibodies that were each titrated individually for their optimal stain index. PBMCs were stained at 10 million cells/mL in 200uL PBS. All PBMCs were incubated for 10 minutes with an amine-reactive viability dye (LIVE/DEAD Aqua, Invitrogen), washed twice, and then stained for 15 minutes at room temperature with combinations of monoclonal antibodies. For ex vivo phenotyping, cells were stained with CD3-AF700 (UCHT1, BD), CD4-PECy5 (RPA-T4, BD), CD8-APC-AF750 (3B5, Invitrogen), CD45RO-PETR (UCHL1, Beckman Coulter), CCR7-BV421 (150503, BD), CXCR5-AF488 (RF8B2, BD), PD-1-PE (EH12.2H7, BioLegend), CD14-V500 (M5E2, BD), and CD19-V500 (HIB19, BD). In vitro phenotyping was performed with combinations of CD3-AF700, CD4-PECy5, CD8-APC-AF750, CD45RO-PETR, CXCR5-AF488, CD14-V500, CD19-V500, ICOS-PE (DX29, BD), CD40L-PE (TRAP1, BD) and PD-1-BV421 (EH12.2H7, BioLegend). All PBMCs were washed twice after staining, fixed with 2% paraformaldehyde, and analyzed on a BD LSR Fortessa (BD Biosciences) at the VMC Flow Cytometry Shared Resource.
Flow cytometry data was analyzed using BD Biosciences FACSDiva Software. In all experiments, forward and side scatter were used to identify lymphocytes and from that population non-viable, CD14+, CD19+, CD8+ cells were excluded from further analysis (Fig. S1).

Nicholas K.J., Flaherty D.K., Smith R.M., Sather D.N, & Kalams S.A. (2017). Chronic HIV-1 infection impairs superantigen-induced activation of peripheral CD4+CXCR5+PD-1+ cells, with relative preservation of recall antigen-specific responses. Journal of acquired immune deficiency syndromes (1999), 74(1), 72-80.

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Murine Spleen and Bone Marrow Immunophenotyping

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The spleen samples were obtained from NHD13 and WT mice, and were stored in 2 ml PBS and gently ground. The cell suspension was acquired after being filtered, centrifuged and after hemolysis (Hemolysin, Becton Dickinson Company, USA). Finally, 1 × 10⁶ cells were immunostained with rat-anti-mouse monoclonal CD4-FITC, CXCR5-APC, PD-1-PE, OX40-PE, ICOS-PE and CD40L-PE (BD company, USA) respectively, and analyzed by FCM (BD Accuri C6, BD company, USA). Meanwhile, BM samples were acquired from the femur of the mice. After being filtered, 1 × 10⁶ cells were stained with CD4-FITC, CXCR5-APC, PD-1-PE, OX40-PE, ICOS-PE and CD40L-PE respectively, and analyzed by FCM. The data was analyzed by BD Accuri C6 software (BD Company, USA).

Jiang H., Cui N., Yang L., Liu C., Yue L., Guo L., Wang H, & Shao Z. (2017). Altered follicular helper T cell impaired antibody production in a murine model of myelodysplastic syndromes. Oncotarget, 8(58), 98270-98279.

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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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