The tumor tissues were ground and filtered to obtain cell suspensions. The cells were collected by centrifugation at 1000 RPM for 5 min and washed with PBS three times, and the supernatant was removed by centrifugation. Then cells were fixed via a cell fixation solution for 30 min and permeabilized by 1% Triton-100 at 37 °C for 10 min, followed by antibody incubation (CD3-PE: 0.25 μg/Test; CD4-FITC: 0.25 μg/Test; CD8a-FITC: 1 μg/Test; CD25-Cy7: 0.25 μg/Test; Foxp3-APC: 1 μg/Test, BioLegend, San Diego, CA, USA) at 37 °C in the dark for 30 min. The cell phenotypes were detected by flow cytometry after 500 μL PBS was added.
Foxp3 apc
Manufactured by BioLegend
Sourced in United States
Foxp3-APC is a fluorescently labeled antibody that binds to the Foxp3 transcription factor, which is a key regulator of regulatory T cells (Tregs). This antibody can be used for the identification and characterization of Tregs in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd25 pe, by BioLegend (3 mentions)
Cd4 fitc, by BioLegend (2 mentions)
Cd3 pe, by BioLegend (1 mentions)
Cd8a fitc, by BioLegend (1 mentions)
Foxp3 fix perm buffer set, by BioLegend (1 mentions)
Il 4 bv605, by BioLegend (1 mentions)
Apc anti mouse cd11c, by BioLegend (1 mentions)
Anti mouse cd4 pe, by BD (1 mentions)
Anti mouse cd86 fitc, by BD (1 mentions)
Mhc 2 pe cy7, by BD (1 mentions)
Cd4 bv605, by BioLegend (1 mentions)
Il 17 apc cy7, by BioLegend (1 mentions)
Cd11c apc, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Pd l1 pe, by BD (1 mentions)
Cd4 pe cy7, by BioLegend (1 mentions)
Cd3 alexa 488, by BioLegend (1 mentions)
Corn oil, by Merck Group (1 mentions)
Ch223191, by Merck Group (1 mentions)
Fixation permeabilization kit, by BD (1 mentions)
8 protocols using foxp3 apc
1
Tumor Immune Cell Phenotyping
2
Characterization of Immune Cell Activation
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In order to examine activation status of the cells, BMDCs were treated with Duolac ATP or LPS for 24 h at 37°C. The cells were stained with anti-mouse CD86-FITC, PD-L1-PE, MHC II-PE-cy7, CD11c-APC (all from BD Biosciences) for 20 min at 4°C in the dark. To test the increase of Treg, CTV labeled CD4+ T cells cultured with Duolac-treated BMDCs for 3 days were stained with anti-mouse CD4-PE (BD Biosciences). After surface staining, CD4+ T cells were fixed and stained with anti-mouse Foxp3-APC mAb (BioLegend, Dedham, MA, United States) using FOXP3 Fix/Perm Buffer Set (BioLegend). In vivo examination, single cells from mLNs and PP were isolated from AD mice. Population changes of DCs and Tregs were examined as aforementioned. To analyze for subpopulation of Th cells, total mLN and PP cells were stimulated with PMA and ionomycin (Sigma-Aldrich) in the presence of brefeldin A for 4 h. After the stimulation, the cells were stained with appropriate combination of anti-mouse CD11c-APC, CD4-bv605, Foxp3-APC, IFN-gamma-PE, IL-4-bv605, and IL-17-APC-cy7 mAb (all from Biolegend). The cells were washed and the expression was examined using a FACSCanto II (BD Biosciences). All flow cytometric data acquired were analyzed with FlowJo software (Tree Star, Ashland, OR, United States).
3
Immunomodulatory Effects of TCDD and FICZ
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TCDD was kindly provided by Dr. Steve Safe (Institute of Biosciences & Technology, Texas A&M Health Sciences Center, College Station, Texas). FICZ was purchased from Enzo Life Sciences (Farmingdale, NY). Both TCDD and FICZ dissolved in DMSO were used for in vitro studies and dissolved in corn oil used for in vivo studies. mBSA and corn oil were purchased from Sigma-Aldrich (St. Louis, MO). RPMI 1640, L-Glutamine, penicillin-streptomycin, HEPES, PBS, and FBS were purchased from Invitrogen Life Technologies (Carlsbad, CA). AhR antagonist (CH223191) was purchased from Sigma-Aldrich, N.C. Fluorophore labeled monoclonal antibodies (mAbs) such as CD3-Alexa 488, CD4-PE/Cy7, CD8-Alexa 700, Foxp3-APC, IL-17-FITC, TGF-β1- PerCP-Cy5.5, IL10-PE, and Helois- PE–Dazzle, used for the flow cytometry, were purchased from Bio Legend (San Diego, CA) and Thermo fisher (Grand Island, NY). For intracellular staining, we used fixation/permeabilization kits from BD Biosciences for IL-17, and Foxp3 fix/perm buffer from Thermo fisher (Grand Island, NY).
4
Foxp3 Nuclear Co-localization Assay
5
Antibody-mediated Immune Profiling
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Therapeutic anti-CTLA-4 (clone 9H10) antibody and isotype control antibody was purchased from BioXcell (cat: BE0131 and BE0087). Antibodies used for flow cytometry were purchased from the following sources (dilutions are indicated in parentheses): eBioscience (CD45.2 Alexa Fluor 700, cat: 56-0454 (1:200), CD3 PE-Cy7, cat: 25-0031 (1:200), CD4 ef450, cat: 48-0041 (1:200), CD4 APC-efluor780, cat: 47-0041 (1:400), CD8 PerCP-efluor710, cat: 46-0083 (1:200), CD11b APC-efluor 780, cat: 47-0112 (1:600), ICOS PE, cat: 12-5985 (1:200), ICOSL PE, cat: 12-5985 (1:200), CTLA-4 PE, cat: 12-1522 (1:200), NK1.1 PE, cat: 12-5941 (1:200), IFNγ PE, cat: 12-7311 (1:200), FoxP3 Alexa Fluor 700, cat: 56-5773 (1:100), FoxP3 APC, cat: 17-5773 (1:200), GATA-3 PE, cat: 12-9966 (1:100), RORγT PerCP-efluor710, cat: 46-6981 (1:100), Tbet PE-Cy7, cat: 25-5825 (1:100), EOMES efluor 450, cat: 48-4875 (1:100), PD-1 PE-Cy7, cat: 25-9985, (1:200)), Biolegend (CD3 BV570, cat: 100225 (1:100), CD11b BV570, cat: 101233 (1:50), Bcl-6 Alexa 594, cat: 648308 (1:50)), Invitrogen (Granzyme B PE-Texas Red, cat: GRB17 (1:125), Granzyme B APC, cat: GRB05 (1:125)) and BD Pharmingen (Ki-67-Alexa Fluor 488, cat: 561165 (1:50), CXCR5-biotin, cat: 551960 (1:100)).
6
Dioscin Modulates Regulatory T Cells
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Dioscin (purity ≥ 98%; lot number: B21176) was purchased from Shanghai Yanye Bio-Technology Co., Ltd (Shanghai, China). Mouse IL-10, IL-35 and TGF-β ELISA KIT were purchased from Sangon Biotech (Shanghai, China). CD4 + CD25+ Regulatory T Cell Isolation Kit was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). The fluorophore-conjugated monoclonal antibodies: CD4-FITC, CD25-PE, Foxp3-APC, CD127-APC, CD152-APC, CD357-PerCP, CD39-PerCP, CD73-FITC and Perforin-APC were from Biolegend (San Diego, CA, USA).
7
Stimulation-Induced CD4+ T Cell Analysis
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CD4 + T cells were harvested before and after stimulation. Cell surface markers were stained with the indicated labeled antibodies against the indicated cell surface antigens. Cells were prepared in heparinized tubes by Ficoll-Paque density gradient centrifugation and were then analyzed on a FACSCanto (Invitrogen, Carlsbad, CA, United States) using FlowJo software (Tree Star) according to the manufacturer’s instructions. The following antibodies were used for flow cytometry to analyze the cell types and cytokine production: PE-CD4, Foxp3-APC, CD25-PE and FITC-IL-17A (BioLegend, CA, United States). FITC-, PE- and APC labeled mouse IgG antibodies were utilized as isotype controls (BioLegend, CA, United States).
8
Rat Acetylcholine Receptor Autoantibody Assay
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The R97-116 peptide derived from the rat (AChRα) subunit was used as an immunogen. The peptide (DGDFAIVKFTKVLLDYTGHI) was obtained from Bankpeptide Technology Co. (Hefei, China) (MW: 2252.57 Da, purity >95%). Mycobacterium tuberculosis H37RA powder was purchased from DIFCO (Franklin Lakes, NJ, USA). Freund’s adjuvants were obtained from Sigma (St. Louis, MO, USA.). Rat AChR-Ab ELISA test kits (QS41981) were purchased from Qisong Biotechnology Co. (Beijing, China). Sotrastaurin (AEB-071) was produced by GLPBIO (Montclair, CA, USA). Antibodies (anti-rat CD4-FITC, CD25-PE, and Foxp3-APC), fixation buffer, true-nuclear transcription factor buffer sets, intracellular staining permeabilization buffer, and leukocyte cell activation cocktail were purchased from Bio Legend, Inc. Erythrocyte Lysis Buffer and rat interleukin (IL)-17A-APC were purchased from eBioscience, Inc. (San Diego, CA, USA) Azathioprine (AZP) and 0.9% saline (NS) were provided by the Pharmacy Department of the 8th Medical Center of Chinese PLA General Hospital. RPMI-1640 was prepared in-house. The optical density of the test sample and the standard was determined with a Bio-Kinetics microplate reader at a wavelength of 450 nm. Flow cytometry was performed on a BD LSRII flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).
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