FCM analysis was performed to analyze the expression of marker molecules, including CD11c, DEC205, CD4, CD8, CD45, CD25, Foxp3, and Gr-1, on the cell membranes, as well as intracellular cytokines, including IFN-γ, TNF-α, and IL-2.24 (link) Cells stained with fluorescent dyes, such as CFSE and DiI, were also analyzed by FCM. The cells isolated from various tissues were incubated with the corresponding fluorescent dyes or monoclonal antibodies conjugated to FITC, PE, PerCP-Cy5.5, or APC for 30 min at 4°C in 1:100–150 dilutions. The following monoclonal antibodies were purchased from eBioscience (San Diego, California, USA) or BD Biosciences: anti-DEC205, anti-CD11c, anti-CD45, anticalreticulin (anti-CRT), anti-CD8, anti-IFN-γ, anti-TNF-α, anti-CD4, anti-CD25, anti-FOXP3, anti-Gr-1, anti-CD206, and anti-F4/80. Before intracellular cytokine staining, 2×106 splenocytes were stimulated with tumor lysates (5 µg/mL) in culture medium with 10% fetal calf serum and 2 µg/mL brefeldin A (BD Bioscience) for 6 hours at 37°C. The intracellular cytokines in splenocytes or TILs were stained using the Cytofix/Cytoperm kit (BD Bioscience) as per the manufacturer’s protocol. The stained cells were detected by FCM (CyFlow Cube 6; Sysmex, Japan), and the resultant data were analyzed to capture the FCM images with FlowJo software version 10 (Tree Star, Ashland, Oregon, USA).
Anti foxp3
Manufactured by BD
Sourced in United States
Anti-Foxp3 is a laboratory reagent used for the detection and analysis of the Foxp3 protein, which is a transcription factor essential for the development and function of regulatory T cells. It is commonly used in flow cytometry, immunohistochemistry, and other research applications to identify and study regulatory T cell populations.
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Lab products found in correlation
Anti cd8, by BD (11 mentions)
Anti cd4, by BD (11 mentions)
Anti cd3, by BD (11 mentions)
Anti ifn γ, by BD (6 mentions)
Anti il 17a, by BD (4 mentions)
Anti cd62l, by BD (4 mentions)
Anti cd45, by BD (3 mentions)
Anti cd11c, by BD (3 mentions)
Anti cd69, by BD (3 mentions)
Anti granzyme b, by BD (3 mentions)
Anti cd44, by BD (3 mentions)
Anti il 4, by BD (3 mentions)
Facscanto 2, by BD (3 mentions)
Phorbol myristate acetate (pma), by Merck Group (3 mentions)
Ionomycin, by Merck Group (3 mentions)
Anti cd4, by BioLegend (3 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Anti tnf α, by BD (2 mentions)
Facscanto 2 cytometer, by BD (2 mentions)
Dnase, by Merck Group (2 mentions)
28 protocols using anti foxp3
1
Comprehensive Immunophenotyping by Flow Cytometry
2
Evaluating T Reg Cells in Asthmatic Mice
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To evaluate the recruitment of Treg induced by ASCs treatment, the LLN cells were cultured in plate-coated anti-CD3 for 3 hours from the LLNs of OVA-induced asthmatic mice and ASC-treated asthmatic mice. The cells were stained with anti-CD25-APC (0.2 mg/mL), anti-CD4-FITC (0.5 mg/mL), and anti-Foxp3 (0.2 mg/mL) in accordance with the manufacturer's recommendations (BD Biosciences, San Jose, CA).
To stain intracellular IFN-γ and IL-4, the LLN cells were first stained for CD4, subsequently fixed, permeabilized using Cytofix/Cytoperm Kit (BD Biosciences), and incubated with PE-cy7-conjugated anti-IFN-γ or PE-conjugated anti-IL-4. Fluorescence was measured using a FACS CantoII cytometer (BD Biosciences) equipped with Canto software (BD Biosciences).
To stain intracellular IFN-γ and IL-4, the LLN cells were first stained for CD4, subsequently fixed, permeabilized using Cytofix/Cytoperm Kit (BD Biosciences), and incubated with PE-cy7-conjugated anti-IFN-γ or PE-conjugated anti-IL-4. Fluorescence was measured using a FACS CantoII cytometer (BD Biosciences) equipped with Canto software (BD Biosciences).
3
Multiparametric PBMC Phenotyping
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PBMCs were isolated by Histopaque-1077 (Sigma Aldrich, USA) density gradient centrifugation and, after gating for CD3 positivity, were stained with anti-CD4 PE, anti-CD25FITC (BD Biosciences, USA), anti-CD69 and anti-FoxP3 for FACS fluorescence cytometry. After gating for Lin-negativity, the isolated PBMCs were also stained for the myeloid dendritic markers CD11c and CD86PE (eBioscience, USA). The data were analyzed with a Beckman Coulter flow cytometer.
4
Tumor Immune Profiling by Flow Cytometry
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The treated mice were euthanized on day 18 following tumor inoculation, and tumors were harvested. The tumors were dissected into fragments by cutting and digested by collagenase (0.5 mg/mL, MilliporeSigma) and DNase (1 μg/mL, MilliporeSigma) at 37°C for 45 minutes. The digested samples were then filtered through a 70 μm cell strainer and washed with PBS buffer. The cell pellets were incubated with RBC lysis buffer to lyse the RBCs. The cell suspensions were stained for the intracellular and extracellular protein markers of interest, and the stained samples were assessed on a flow cytometer (BD biosciences) along with CellQuest Pro software. The following staining antibodies used: anti-CD3, anti-CD4, anti-CD19, anti-Foxp3, anti-CD8, anti–granzyme B, anti–active caspase-3, and anti–IFN-γ (all from BD Biosciences).
5
Multi-Parametric Flow Cytometry Analysis
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Labelled anti-CD4 (GK1.5, 1:100), anti-CD8 (53-6.7, 1:100), anti-CD25 (PC61, 1:100), anti-CD44 (IM7.8.1, 1:100), anti-CD62L (MEL14, 1:100), anti-FOXP3 (FJK-16s, 1:100), anti-GATA3 (L50-823, 1:100), anti-IL-2 (JES6-5H4, 1:100), anti-IL-4 (BVD4-1D11, 1:100), anti-IL-17A (TC11-18H10, 1:100), anti-IFN-γ (XMG1.2, 1:100) and specific isotype-matched control antibodies (1:100) were from BD Biosciences, eBioscience or Miltenyl Biotec. Anti-USP21 (G-17, 1:1,000), Anti-Ubiquitin (P4D1, 1:1,000), anti-GATA3 (HG3-31, 1:1,000) was from Santa Cruz Biotechnology. anti-FOXP3 (eBio7979, 1:1,000) was from eBioscience.
6
Isolation and Analysis of Immune Cells
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Single-cell suspensions from spleen and lymph nodes were prepared using a 40 µm nylon mesh. Blood was collected in Alsevers and subjected to erythrolysis. CNS-infiltrating cells and sorted microglia and T cells were purified as described below. All antibodies were obtained from BioLegend (Uithoorn, The Netherlands) unless otherwise indicated and directly conjugated to a fluorochrome: anti-CD3ε (17A2), anti-TCRβ (H57-597), anti-CD4 (RM4-5, BD Biosciences), anti-CD11b (M1/70), anti-CD25 (PC61), anti-CD45.2 (104), anti-Ly6C (HK1.4), anti-Ly6G (1A8), and anti-FoxP3 (FJK-16s). Stainings were performed as previously described (12 (link)). Analysis was carried out using a FACSCanto II device (BD Biosciences) in combination with FlowJo software (Treestar, Ashland, OR, USA).
7
Flow Cytometry Analysis of Regulatory T Cells
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Cells were incubated with antibodies at the optimized concentrations for 30 minutes at 4°C. The samples were centrifuged, washed twice with PBS/1% fetal bovine serum, and analyzed using a FACSCanto (Becton Dickinson) flow cytometer. For flow cytometry analysis of regulatory T cells, anti-CD4, anti-CD8, anti-CD25, anti-GITR, anti-CD62L, and anti-Foxp3 antibodies in various fluorochrome combinations were purchased from BD Biosciences (San Diego, CA).
8
Monocyte Isolation and FOXP3 Analysis
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Monocytes were isolated from peripheral blood as described previously. After incubation with anti-CD4 and anti-CD45 (BD Biosciences, San Diego, USA) at 4 ˚C for 30 min in the dark, the cells were stained with anti-FOXP3 (BD Biosciences) and then analyzed by flow cytometry.
9
Quantifying Regulatory T Cells in Lungs
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For these studies, bronchoalveolar cells (1 × 106/mL) were incubated with anti‐mouse CD3 FITC conjugate and B220 PE (R&D Systems, San Diego, California) for 30 min at 4°C. Cells were washed twice with cold PBS and resuspended in PBS; 10.000 events were acquired using a Becton Dickinson FACScan, and the data were analyzed using WinMIDI software. Cell populations were identified according to criteria described by Van Rijt and colleagues 25 . To assess Treg cells were used antibodies anti‐FOXP3 (BD Biosciences Pharmingen), anti‐CD4 and anti‐CD25 (Santa Cruz Biotechnology). Cells from lymph nodes were fixed with paraformaldehyde 2% for 10 min at 37°C and permeabilized in ice with 90% methanol for 15 min. Before labeling, the cells were washed three times in HBSS and incubated with antibodies for 20 min. At the end of incubation, cells were washed and suspended in HBSS for further analyses on flow cytometer.
10
Analyzing Th17, Th1, and Treg Cells in EAE Mice
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Mouse-specific anti-CD3 (Pacific Blue conjugated, clone 500A2, catalog no. 558214), anti-CD4 (PerCP conjugated, clone RM4-5, catalog no. 553052), anti-CD25 (Phycoerythrin/Cy7, clone PC61, catalog no. 561780), anti-FoxP3 (Alexa Fluor 488 conjugated, clone MF23, catalog no. 560403), anti-IL17A (Alexa Fluor 647 conjugated, clone TC11-18H10, catalog no. 560184), and IFNγ (PE conjugated, clone XMG1.2, catalog no. 554412) mAbs were all purchased from BD Biosciences.
For intracellular staining total splenocytes were isolated 37 days after EAE induction and cells were re-stimulated for 3 days with increasing amounts of MOG35-55. After that cells were stimulated for 4 h with PMA (50 ng/ml) and ionomycin (500 ng/ml) in the presence of GolgiPlug (1:1,000, BD Pharmingen). Cell were stained with surface markers (CD3, CD4, CD25), permeabilized using the eBioscience™ Foxp3/Transcription Factor Staining Buffer Set and stained for IL-17, IFNg, and FoxP3.
Samples were acquired on BD FACS Canto II flow cytometer and analyzed with FlowJo software.
The absolute count of Treg, Th1, and Th17 cells was measured as % singlets on 20,000 cells X % Treg or Th1 or Th17 on singlets/100.
For intracellular staining total splenocytes were isolated 37 days after EAE induction and cells were re-stimulated for 3 days with increasing amounts of MOG35-55. After that cells were stimulated for 4 h with PMA (50 ng/ml) and ionomycin (500 ng/ml) in the presence of GolgiPlug (1:1,000, BD Pharmingen). Cell were stained with surface markers (CD3, CD4, CD25), permeabilized using the eBioscience™ Foxp3/Transcription Factor Staining Buffer Set and stained for IL-17, IFNg, and FoxP3.
Samples were acquired on BD FACS Canto II flow cytometer and analyzed with FlowJo software.
The absolute count of Treg, Th1, and Th17 cells was measured as % singlets on 20,000 cells X % Treg or Th1 or Th17 on singlets/100.
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