Icos pe

Manufactured by BD

Sourced in United States

ICOS-PE is a flow cytometry instrument designed for the detection and analysis of cell surface markers. It utilizes the principle of flow cytometry to measure the fluorescence intensity of labeled cells as they pass through a laser beam.

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Anti-ICOS–PE (11 citations) PE)-conjugated anti-ICOS (2 citations)

6 protocols using icos pe

Multiparametric Flow Cytometry Analysis

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Multi-color flow cytometry analysis was performed on PBMCs from all time points by staining for 30 minutes at 4°C with CD3-V450, CD8-FITC or APC, ICOS-PE, HLA-DR-PerCP-Cy5.5, CD25-PE-Cy7, CD45RA-PerCP-Cy5.5, CD62L-FITC, CD127-V450, PD-1-PE, Tim-3-AF700, CD4-APC-Cy7 (BD Biosciences, San Jose, CA), CCR7-PE-Cy7 (R&D Systems, Minneapolis, MN), CTLA-4-FITC (LSBio, Seattle, WA) and FoxP3-APC (eBioscience, San Diego, CA) for T cells. For natural killer (NK) cells, CD3-V450, CD16-APC-Cy7, CD56-PE-Cy7 and Tim-3-AF700 (BD) were used. For myeloid-derived suppressor cells (MDSCs), CD33-PE, CD11b-APC-Cy7, HLA-DR-PerCP-Cy5.5, CD14-V450 and CD15-APC (BD) were used. 1×10⁵ cells were acquired on an LSRII (BD), and data was analyzed using FlowJo software (Tree Star Inc., Ashland, OR). The appropriate isotype controls were used, and dead cells were excluded from the analysis.

Jochems C., Tucker J.A., Tsang K.Y., Madan R.A., Dahut W.L., Liewehr D.J., Steinberg S.M., Gulley J.L, & Schlom J. (2014). A Combination Trial of Vaccine Plus Ipilimumab in Metastatic Castration-Resistant Prostate Cancer Patients: Immune Correlates. Cancer immunology, immunotherapy : CII, 63(4), 407-418.

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Multiparametric Immune Phenotyping of Cells

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Cells were thawed and cultured in AIM-V culture medium with 5% heat-inactivated human serum in 24-well plates at a concentration of 3–5 × 10⁶ cells/ml. Plates were incubated in a humidified incubator at 37°C, with 5% CO₂. After 48 hours of resting, cells were harvested and washed in FACS buffer before surface staining. For exhaustion profile analysis, cells were stained at 4°C for 30 min., washed, and, for tubes only intended for surface staining, resuspended in FACS buffer. For tubes intended for intracellular staining, cells were permeabilized using BD Bioscience Cytofix/CytopermTM Kit according to manufacturer's instructions. The following antibodies were used: CD4-PerCP, CD57-FITC, CD27-PE, CD56-PE-Cy7, CD28-APC, CD8-AmCyan, ICOS-PE, BTLA-PE, CTLA-4-APC, PD-1-PE-Cy7 (all from BD Bioscience, San Jose, CA, USA), Near Infra Red dead cell marker (Dako), LAG-3-FITC (LifeSpan Biosciences), TIM-3 (eBioscience), CD107a; PECy7-conjugated IFN-c; PerCP-conjugated CD8; APC-conjugated CD4, TNF-a; APCCy7-conjugated CD3. Cells were resuspended in FACS buffer prior to acquisition, and the cells were acquired using a BD FACSCanto II flow cytometer. A minimum of 100 000 events were recorded per sample. Analysis was performed with the BD FACSDiva Software (BD Bioscience,)

Bjoern J., Lyngaa R., Andersen R., Rosenkrantz L.H., Hadrup S.R., Donia M, & Svane I.M. (2017). Influence of ipilimumab on expanded tumour derived T cells from patients with metastatic melanoma. Oncotarget, 8(16), 27062-27074.

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Flow Cytometry Analysis of PBMC Responses

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Frozen PBMCs from vaccinated participants were thawed and then analyzed by flow cytometry both ex vivo and after 18 h of stimulation at 37°C with anti-CD28 and anti-CD49d (1 μg/ml each, BD Biosciences), A/California/7/2009 (H1N1) subunit vaccine antigen (1 μg/ml, Novartis Vaccines & Diagnostics), or Staphylococcus enterotoxin B (SEB) (1 μg/ml, Sigma), in the presence of Brefeldin A (5 μg/ml, Sigma) as previously described [18 (link), 25 (link)]. Cells were stained ex vivo with Live/Dead Yellow (Invitrogen), fluorochrome-conjugated antibodies: CD3-PE-Texas Red (SK7), CD4-APC-Horizon 7 (SK3), ICOS-PE (ISA-3), CXCR5-FITC (RF8B2), CXCR3-PE-Cy5 (1C6), CCR6-Brillant Violet 421 (11A9), PD1-Brilliant Violet 785 (EH12.2H7), CD19-APC (SJ25C1), CD20-PerCP-Cy5.5 (L27), CD27-PE (L128), CD38-Alexa fluor 700 (HIT2) (BD Biosciences), CD8-Horizon V500 (RPA-T8) (Biolegend), CD45RA-PE-Cy7 (HI100) (eBioscience). H1N1-specific CD4⁺ T cells were analyzed for intracellular production of IL-21 with anti-IL-21-APC (3A3-N2.1) (BD Biosciences) [18 (link), 25 (link)]. Stained cells were acquired on a BD LSR Fortessa special order flow cytometer (BD Biosciences).

Spensieri F., Siena E., Borgogni E., Zedda L., Cantisani R., Chiappini N., Schiavetti F., Rosa D., Castellino F., Montomoli E., Bodinham C.L., Lewis D.J., Medini D., Bertholet S, & Del Giudice G. (2016). Early Rise of Blood T Follicular Helper Cell Subsets and Baseline Immunity as Predictors of Persisting Late Functional Antibody Responses to Vaccination in Humans. PLoS ONE, 11(6), e0157066.

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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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Comprehensive Immune Profiling in SLE and RA

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PBMCs from patients with SLE or RA and HC were collected and analyzed immediately for the molecular phenotypes of lymphocytes. The antibodies used for the surface marker analysis include anti-human CD3-ECD (Beckman Coulter, USA), CD4-Percp-Cy5.5, CD8-PE-Cy7, PD1-FITC, TIGIT-APC, ICOS-PE, TIM3-BV421, CD19-V500, CD3-FITC, CD8-V500, CD25-APC, HLA-DR- PE-Cy7, CD69-V450, and CD127-PE (BD Biosciences, USA). Briefly, 50 μl of cells was incubated with appropriate antibodies on ice in the dark for 30 min, followed by washing in PBS. All the samples were analyzed with a Navios flow cytometer (Beckman Coulter) and Kaluza analysis software (Beckman Coulter).

Zhou H., Li B., Li J., Wu T., Jin X., Yuan R., Shi P., Zhou Y., Li L, & Yu F. (2019). Dysregulated T Cell Activation and Aberrant Cytokine Expression Profile in Systemic Lupus Erythematosus. Mediators of Inflammation, 2019, 8450947.

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