Lung samples of mice, mock-inoculated or inoculated with WT-rHMPV or mutants, were harvested at days 5 and 7 p.i. Total lung cells were prepared, as previously described25 (link),26 (link). Isolated cells were incubated with anti-FcγRII/RcγRIIImAb (24G2, BD Biosciences, San Jose, CA, USA) followed by cell-surface marker staining. For cell-surface marker staining, isolated cells were stained with the following antibodies: anti-CD11c in combination with anti-CD80, anti-CD86, or anti-MHCII (all from BD Pharmingen, San Jose, CA, USA) for DCs and anti-CD4 or anti-CD8 in combination with anti-CD3 (all from BD Pharmingen) for T cells. Samples were stained at 4 °C in PBS with 1% FBS and analyzed with a BD FACSCanto flow cytometer equipped with BD FACSDiva software (both from Becton Dickinson Immunocytometry Systems, Franklin Lakes, NJ, USA). The analysis was performed using Cyflogic (Cyflo Ltd, Turku, Finland).
Anti cd80
Manufactured by BD
Sourced in United States
Anti-CD80 is a laboratory reagent used in flow cytometry applications to detect the presence of CD80 (also known as B7-1) on the surface of cells. CD80 is a costimulatory molecule expressed on antigen-presenting cells that plays a role in the activation of T cells. The Anti-CD80 product can be used to identify and quantify cells expressing CD80 in various research and diagnostic settings.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd86, by BD (33 mentions)
Anti cd11c, by BD (22 mentions)
Anti cd40, by BD (10 mentions)
Anti cd4, by BD (8 mentions)
Anti mhcii, by BD (7 mentions)
Anti cd8, by BD (7 mentions)
Anti cd3, by BD (7 mentions)
Anti cd11b, by BD (7 mentions)
Anti cd83, by BD (7 mentions)
Anti hla dr, by BD (6 mentions)
Facscanto 2, by BD (6 mentions)
Anti ifn γ, by BD (5 mentions)
Anti pd l1, by BD (3 mentions)
Anti mhc class 2, by BD (3 mentions)
Facscalibur flow cytometer, by BD (3 mentions)
Anti f4 80, by BD (2 mentions)
Anti cd45, by BD (2 mentions)
Diva software, by BD (2 mentions)
Cellquest software, by BD (2 mentions)
Facscanto, by BD (2 mentions)
43 protocols using anti cd80
1
Immunophenotyping of Lung Cells in HMPV Infection
2
Investigating GIFT4-Modulated B-Cell Interactions
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PBMC from subjects with CLL were stimulated with GIFT4 protein or GM-CSF and IL-4 (2 ng/ml) for 5 days. The cells were harvested and stained with APC-conjugated anti-human CD19 and PE-conjugated anti-human CD5 antibodies; or APC-conjugated anti-human CD3, then subjected to flow cytometry (FACS) on a BD FACSCanto II flow cytometer. Phenotype of GIFT4-CLL cells was profiled by FACS with a panel of B cell antibodies including anti-CD80 and CD86 (BD, San Diego, CA, USA). For analysis of cell–cell interaction between GIFT4-CLL cells and T cells, CFSE-labeled autologous T cells were co-cultured with GIFT4-CLL cells pre-treated with anti-human CD80 or CD86 neutralizing antibodies or isotype control (1 μg/ml) (BioLegend) for 5 days. T cell division was determined by FACS. For intracellular staining of IFN-γ, Granzyme B, and perforin, T cells were fixed and permeabilized with BD Cytofix/Cytoperm™ solution, followed by staining with PE-conjugated anti-human IFN-γ, granzyme B, perforin antibodies (BD). Alternatively, circulating human T cells in the peripheral blood of NSG immune deficient mice adoptively transferred with PBMC from CLL patients were profiled and counted by FACS, and analyzed with FlowJo 9.1 software.
3
Multiparametric Flow Cytometry of Tumor-Infiltrating Immune Cells
4
Intestinal Macrophage Phenotyping and Activation
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The presence of MΦ in the intestinal lamina propria of CDA or HC subjects and the phenotype of monocytes derived MΦ was evaluated by flow cytometry as follow: MΦ were stained with antibodies anti-CD80 (BD bioscience) and CD206 (BD bioscience) to evaluate the percentages of classically activated pro-inflammatory (M1) and alternatively activated anti-inflammatory (M2) MΦ, respectively; CD 68 antibody was used as a pan marker for MΦ. Activation of IFNγR and pSTAT1 on monocytes derived MΦ was evaluated using IFNγR (Thermofisher) and intracellular pSTAT1 (Ser767) (Abcam) antibodies after methanol permeabilization. Data were acquired with BD FACS Calibur flow cytometer instrument and analyzed with BD CellQuestPro and FlowJo softwares.
5
Characterizing Bacterial Infection in Macrophages
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To assess bacterial infection in macrophages, HMDMs were harvested after adding non-enzymatic cell dissociation solution (Sigma-Aldrich, Oakville, ON, Canada) for 30 mins at room temperature. Cells were washed with PBS and prepared in FACS tubes. For surface staining, we used the following specific monoclonal antibodies provided from BD Biosciences for phenotypic analysis of human HMDMs: anti-CD3 (BV605), anti-CD14 (BV650), anti-CD80 (PE Cy7), anti-CD209 (PE) and anti-CD64 (PE). Finally, the viability marker 7-aminoactinomycin D or 7-AAD (ThermoFisher Scientific, St. Laurent, QC, Canada) was used to exclude dead cells from analyses. Afterwards, cells were fixed with 4% paraformaldehyde solution and then washed. For data analysis, samples were analyzed by flow cytometry with a BD LSRII Fortessa flow cytometer and DIVA software (BD). Viable gated cell singlets were analyzed for each sample and the percentages of mCherryhigh CFT073 containing CD14+ HMDMs were determined. For Imaging flow cytometry, the same procedures were applied while cells were prepared in Eppendorf tubes. For surface staining, we used CD14-V450 and CD80-APC H7 for phenotypic gating of HMDMs. Samples were acquired using the Image Stream X MKII flow cytometer and analyzed with IDEAS software (Amnis) and representative images of singlets CD14+ HMDMs were generated expressing GFPhigh CFT073 bacterial cells.
6
Multiparametric Phenotyping of Dendritic Cells
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The expressions of HLA, costimulatory molecules and GFP to DCs were analyzed by flow cytometry (FACS Caliber, BD Biosciences). The cells were washed once in PBS and stained for 30min with PE-conjugated anti-HLA-A, -B, -C, anti-4-1BBL, anti-CD80, anti-CD83, and anti-CD86, FITC-conjugated anti-HLA-DR, -DP, -DQ (BD Biosciences, USA). And the cells were washed followed fixed using 1% paraformaldehyde solution. Approximately more than 10,000 cells were acquired and the data were analyzed by using FLOWJO software (Tree Star, USA).
7
Generation of Tolerogenic and Mature Dendritic Cells
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Monocytes were isolated from PBMC of healthy donors using CD14 microbeads (Miltenyi Biotec, Germany). The cells were resuspended in VLE-RPMI 1640 (Biochrom AG, Germany) with 1.5% human serum and seeded at 5 × 106 cells in 25 ml Nunclon™flasks (Nunc, Germany). On day 0, 100 ng/ml GM-CSF and 20 ng/ml IL-4 were added to the cultures. At day 1, 1 ng/ml TGF-β1 and 20 ng/ml IL-10 were added only to tolDC. On the following day, maturation cocktails30 (link) (Table 2 ) were added to tolDC and C5-DC to induce maturation. For immunophenotyping of tolDC and C5-DC, the following antibodies were used: anti-CD14 (clone: M5E2), anti-CD80 (clone: L307.4), anti-CD83 (clone: HB15e), anti-B7-H1 (clone: MIH1), anti-B7-DC (clone: MIH18) (all from BD Biosciences, USA) and anti-CD86 (clone: 2331, Pharmingen). Stained cells were analyzed by using the LSRII (BD Biosciences). Data were processed by using FlowJo software (Tree Star, Ashland OR, USA).
Maturation cocktail for generation of tolDC and C5-DC.
| TolDC | C5-DC | |
|---|---|---|
| GM-CSF (Leukine sargramostim, USA) | 100 ng/ml | 100 ng/ml |
| IL-4 (R&D, USA) | 20 ng/ml | 20 ng/ml |
| IL-1β (R&D, USA) | 10 ng/ml | 10 ng/ml |
| TNF-α (PEPRO-TECH, USA) | 20 ng/ml | 20 ng/ml |
| PGE2 (Sigma, USA) | 250 ng/ml | 1 µg/ml |
| R848 (In vivo Gen) | — | 1 µg/ml |
| IFN-γ (Boehringer Ingelheim, Germany) | — | 5000 U/ml |
| IL-6 (R&D, USA) | 15 ng/ml | — |
| TGF-β1 (PEPRO-TECH, USA) | 1 ng/ml | — |
| IL-10 (PEPRO-TECH, USA) | 20 ng/ml | — |
8
Immune Cell Immunophenotyping Protocol
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Cells were harvested and counterstained immunophenotypically using anti‐CD83, anti‐CD80, anti‐CD86, anti‐CD40 and anti‐CD11c (BD Pharmingen).
9
Immunophenotyping of RCC85#21 Cell Line
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The following fluorescein isothiocyanate (FITC)-conjugated or phycoerythrin (PE)-conjugated mAbs were used for immunofluorescent staining of the RCC85#21 cell line: anti-HLA class I, anti-HLA-DR, anti-CD54, anti-CD80, anti-CD40 and anti-CD86 (BD Pharmingen). In order to stimulate the expression of costimulatory markers, RCC85#21 cells were incubated with IFN-γ for 48 h at the concentration of 500 IU/ml. Cells were washed and resuspended in FACS buffer (phosphate-buffered saline pH 7.2, 0.2% bovine serum albumin, and 0.02% sodium azide) and incubated with fluorochrome-conjugated mAbs for 15 min at 4°C, then washed with the same buffer before flow cytometric analysis. Data were acquired using an EPICS XL flow cytometer (Beckman Coulter, USA) and analysed using WinMDI Version 2.8 software. The area of positivity was determined using an isotype-matched mAb, and a total of 104 events for each sample were acquired. At least 3 experiment for each sample were performed.
10
Flow Cytometry Analysis of Immune Markers
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Flow cytometry was done using the following monoclonal antibodies (mAb) and appropriate isotype controls: anti-HLA class I (W6/32), anti-HLA DR/DP (Q5/13), anti-HLA DR, anti-CD70, anti-CD80 (all BD Biosciences), anti-CD14, anti-CD83 (both Beckman Coulter), anti-CD86 (BD PharMingen), and anti-CCR7 (R&D systems). For intracellular staining of the TAA NKI/beteb (IgG2b; purified antibody) against gp100, and T311 (IgG2a; Cell Marque Corp., Rocklin, CA) against tyrosinase were used. Cells were also stained intracellular with anti-CD40L (Beckman Coulter). For intracellular staining, cells were fixed for 4′ on ice in 4% (w/v) paraformaldehyde (Merck) in PBS, permeabilized in PBS/2%BSA/0.02% azide/0.5% saponin (Sigma-Aldrich) (PBA/saponin), and stained with mAb diluted in PBA/saponin/2%HS, followed by staining with Alexa488-labeled goat-anti-mouse (BD PharMingen). Flow cytometry was performed with FACSCalibur™ flow cytometer equipped with CellQuest software (BD Biosciences).
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