Splenocyte isotype controls or specific antibodies were incubated with normal cells and single staining was performed to reduce differences. Splenocytes were stained with APC-labelled anti-CD4, FITC-labelled CD25, and PE-labelled Foxp3 antibodies (Biolegend). First of all, CD4, CD25 room temperature away from light staining for 30 min, Foxp3 staining before using a film breaker (Elabscience), according to the instructions, Foxp3 room temperature away from light staining for 30 min. Labelled cells were analysed using a FACSCalibur flow cytometer and CellQuest software (BD Biosciences), and the data were analysed with FlowJo 10.8.1 software.
Foxp3 antibody
Manufactured by BioLegend
Sourced in United States
The FoxP3 antibody is a laboratory reagent used to detect and analyze the presence of the FoxP3 (Forkhead box P3) transcription factor. FoxP3 is a critical regulator of the development and function of regulatory T cells (Tregs), which play a crucial role in maintaining immune homeostasis and suppressing autoimmune responses. The FoxP3 antibody can be used in various immunological techniques, such as flow cytometry and immunohistochemistry, to identify and study Tregs.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur flow cytometer, by BD (1 mentions)
Cellquest software, by BD (1 mentions)
Aurora flow cytometer, by Cytek (1 mentions)
Fixation permeabilization kit, by Thermo Fisher Scientific (1 mentions)
40 μm cell strainer, by BD (1 mentions)
Ab20034, by Abcam (1 mentions)
Envision detection kit, by Agilent Technologies (1 mentions)
5 protocols using foxp3 antibody
1
Immunophenotyping of Regulatory T Cells
2
Phenotyping of AIT-responsive PBMCs
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PBMCs of patients at baseline and after 18-month AIT were isolated and stored in liquid nitrogen. Thawed PBMCs were incubated with 40µg/mL HDM allergen for 5 days, then the harvested cells were prepared at a concentration of 1×106 in 100µl FACS staining buffer and were stained with anti-human CD3, CD4, CD279 (PD-1), CD183 (CXCR3), CD185 (CXCR5), CD294 (CRTH2), CD25 and Foxp3 antibodies (Biolegend, USA). Stained cells were acquired with Cytek Aurora flow cytometer (Cytek Biosciences, Fremont, CA, USA) and the data were analyzed with Flowjo software (Treestar, Ashland, OR).
3
Identifying Regulatory T Cells
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Single-cell suspensions from the spleen were prepared by filtering the tissues through 40 μm cell strainers (BD Bioscience). For characterization of Treg cells, lymphocytes were surface-stained with fluorochrome-labeled CD4 and CD25 antibodies and intracellularly stained with the Foxp3 antibody (all from BioLegend). Intracellular staining was performed with a fixation/permeabilization kit, according to the manufacturer’s protocol (eBioscience). The data were collected from BD FACSCalibur and analyzed by FlowJo software (FlowJo, LLC).
4
Immunohistochemical Analysis of FoxP3 in Gastric Cancer
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FoxP3 protein levels were examined by immunohistochemistry (IHC) in 4-μm-thick paraffin sections cut from a single selected block containing neoplastic and non-neoplastic gastric tissues. Samples were routinely treated using a standard staining procedure (EnVision Detection Kit, Dako, Carpinteria, CA, USA) and were subsequently incubated with anti-FoxP3 monoclonal antibody clone Ab20034 (dilution 1 : 250; Abcam, Cambridge, UK), also repeated by monoclonal another FoxP3 antibody (Biolegend, San Diego, CA, USA). For antibody-negative controls, the primary antibodies were substituted with normal rabbit serum. Predominantly cytoplasmic staining was observed in tumour cells and nuclear staining in lymphocytes. Tregs in tumour stroma was counted in 10 high-power field (HPF, at × 400 magnification), Treg count of >25 per HPF was defined as high Treg, and Treg counts of <5 per HPF was defined as low Treg. The presence of FoxP3-positive tumour cells and the quantification of infiltrated FoxP3+ Treg cells were, respectively, calculated by two investigators with no knowledge of the clinical grouped data.
5
Identification and Quantification of Tregs
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For identification and quantification of Tregs, multicolor flow cytometry was used after surface staining of peripheral blood mononuclear cells with specific antibodies. These antibodies include anti-human CD4 and Foxp3. To quantify the number of Tregs after transfection of CD4+ T cells and incubation under Treg skewing conditions, Tregs were identified by surface staining with anti-human CD4+ and intracellular staining with FoxP3 antibody (Biolegend, San Diego, CA, USA). The amount of Tregs was expressed as a ratio of CD4+Foxp3+ T cells as a percentage of CD4+ T cells. Tregs in patients and healthy volunteers were identified after surface staining of PBMCs with monoclonal antibodies specific for anti-human CD4, CD25, and CD127 and intracellular staining with an anti-human FoxP3 antibody. CD4+CD25highCD127lowFoxp3+ cells were defined as Tregs.
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