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Foxp3 clone pch101

Manufactured by Thermo Fisher Scientific
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Foxp3 (clone PCH101) is a laboratory reagent used for the detection and analysis of the Foxp3 protein, a transcription factor expressed in regulatory T cells. It is a tool for research purposes, and its core function is to provide a means to identify and study Foxp3-expressing cells.

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Lab products found in correlation

Anti cd4 fitc clone rpa t4, by Thermo Fisher Scientific (9 mentions) Anti cd25 apc clone bc96, by Thermo Fisher Scientific (9 mentions) Flow staining buffer, by Thermo Fisher Scientific (9 mentions) Cd3 clone sp34 2, by BD (2 mentions) Ionomycin, by Merck Group (2 mentions) Phorbol 12 myristate 13 acetate pma, by Merck Group (2 mentions) Foxp3 fix perm buffer set, by BioLegend (1 mentions) Anti ki67 antibody, by BD (1 mentions) Anti cd3 cd28 coated microbeads, by Miltenyi Biotec (1 mentions) Cd4 clone sk3, by BD (1 mentions) Cd127 clone ebiordr5, by Thermo Fisher Scientific (1 mentions) Lsrii flow cytometer, by Tree Star (1 mentions) Cd25 clone 4e3, by Miltenyi Biotec (1 mentions) Cd4 clone rpa t4, by BioLegend (1 mentions) Cd8 clone rpa t8, by BioLegend (1 mentions) Hla dr clone l243, by Thermo Fisher Scientific (1 mentions) Cd56 clone cmssb, by Thermo Fisher Scientific (1 mentions) Cd25 clone bc96, by BioLegend (1 mentions) Cd25 clone 2a3, by Thermo Fisher Scientific (1 mentions) Foxp3 clone 236a e7, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

PCH101 (14 citations) FOXP3 (PCH101, (8 citations) Anti-FOXP3 (clone PCH101) (7 citations) Anti-FoxP3-PE (clone PCH101; (6 citations) Clone PCH101 (3 citations) FOXP3-FITC (clone PCH101 (3 citations) FOXP3-eF450 (clone PCH101, (2 citations) APC-conjugated anti-FOXP3 (clone PCH101, (2 citations)

16 protocols using foxp3 clone pch101

1

Expansion and Characterization of Human Tregs

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CD4+CD25++CD127−/low putative Tregs, aseptically flow-sorted from peripheral blood lymphocytes (PBL), were expanded using a modification of our previously described protocol (Figure 1) (18 (link)). Briefly, these cells were stimulated with anti-CD3/CD28-coated microbeads (Miltenyi Biotec, Auburn CA, bead: cell ratio of 1:2) on day 0 and cultured in X-Vivo-15 media supplemented as previously described (18 (link)), including 2000 IU/ml of rhIL-2. At days 12 and 24, (20 ) cultures were re-stimulated as on day 0. Treg cultures were pulsed with 100 nM of rapamycin for 48 hours from day 34-36, given our previous results showing that this optimized Treg suppressive activity. (18 (link)). Tregs were then harvested, washed free of rapamycin, magnetic beads removed, and cryopreserved as previously described. (18 (link)) The Treg phenotype was assessed by staining for CD3 (clone SP34-2, BD, San Jose, CA), CD4 (clone SK3, BD), CD25 (clone 4E3, Miltenyi Biotec), CD127 (clone eBioRDR5, eBioscience, San Diego, CA) and FoxP3 (clone PCH101, eBioscience) using the FoxP3 Fix/Perm Buffer Set (BioLegend, San Diego, CA). In some experiments, Tregs were also labeled with an anti-Ki-67 antibody (Clone B56, BD). Data were acquired on an LSR II flow cytometer and analyzed using FlowJo software (Treestar, Ashland, OR). Positively stained cells were identified using appropriate isotype-control antibodies.
Singh K., Stempora L., R. Donald H., Kirk A., Larsen C., Blazar B, & Kean L. (2014). Superiority of Rapamycin over Tacrolimus in Preserving Non-human Primate Treg Half-life and Phenotype After Adoptive Transfer. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 14(12), 2691-2703.
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2

Tregitope Activation of Regulatory T Cells

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Example 3

Human PBMCS were stimulated directly ex vivo for 4 days in the presence of tetanus toxin peptide TT830-844 alone, Tregitope-289 alone, phytohemagglutinin (a mitogenic positive control) alone, or no stimulus. 1×106 cells were stained with anti-CD4-FITC (clone RPA-T4; eBioscience) and anti-CD25-APC (clone BC96; eBioscience) antibodies for 30 minutes on ice in Flow Staining Buffer (eBioscience) and washed twice with buffer. Following cell-surface staining, cells were fixed and permeabilized (eBioscience) and stained intracellulary for Foxp3 (clone PCH101; eBioscience) following manufacturer's protocol. The frequency of FoxP3 positive CD4+/CD25+ T cells under various culture conditions was enumerated by Flowjo analysis software. There were similar increases in CD25 expression in both the Tetanus- and Tregitope-stimulated samples indicating T cell activation by both peptides (FIG. 3; results shown for Tregitope-289). Expression of FoxP3 within the CD4+CD25+ subset, however, differed significantly depending on the stimulus used. Tetanus stimulation led to a 7% decrease in expression of FoxP3, whereas Tregitope stimulation led to a more than two-fold increase in expression, indicating Th and nTreg activation, respectively.

US10925940B2. Regulatory T cell epitopes, compositions and uses thereof (2021-02-23). EpiVax, Inc. [US]. Inventors: Anne De Groot [US], William Martin [US], Daniel S. Rivera [US].
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3

Tregitope Induces FoxP3 Expression

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Example 3

Human PBMCS were stimulated directly ex vivo for 4 days in the presence of tetanus toxin peptide TT830-844 alone, Tregitope-289 alone, phytohemagglutinin (a mitogenic positive control) alone, or no stimulus. 1×106 cells were stained with anti-CD4-FITC (clone RPA-T4; eBioscience) and anti-CD25-APC (clone BC96; eBioscience) antibodies for 30 minutes on ice in Flow Staining Buffer (eBioscience) and washed twice with buffer. Following cell-surface staining, cells were fixed and permeabilized (eBioscience) and stained intracellularly for Foxp3 (clone PCH101; eBioscience) following manufacturer's protocol. The frequency of FoxP3 positive CD4+/CD25+ T cells under various culture conditions was enumerated by Flowjo analysis software. There were similar increases in CD25 expression in both the Tetanus- and Tregitope-stimulated samples indicating T cell activation by both peptides (FIG. 3; results shown for Tregitope-289). Expression of FoxP3 within the CD4+CD25+ subset, however, differed significantly depending on the stimulus used. Tetanus stimulation led to a 7% decrease in expression of FoxP3, whereas Tregitope stimulation led to a more than two-fold increase in expression, indicating Th and nTreg activation, respectively.

US10980867B2. Regulatory T cell epitopes, compositions and uses thereof (2021-04-20). EpiVax, Inc. [US]. Inventors: Anne De Groot [US], William Martin [US], Daniel S. Rivera [US].
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4

Tregitope Induction of Regulatory T Cells

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Example 3

Human PBMCS were stimulated directly ex vivo for 4 days in the presence of tetanus toxin peptide TT830-844 alone, Tregitope-289 alone, phytohemagglutinin (a mitogenic positive control) alone, or no stimulus. 1×106 cells were stained with anti-CD4-FITC (clone RPA-T4; eBioscience) and anti-CD25-APC (clone BC96; eBioscience) antibodies for 30 minutes on ice in Flow Staining Buffer (eBioscience) and washed twice with buffer. Following cell-surface staining, cells were fixed and permeabilized (eBioscience) and stained intracellulary for Foxp3 (clone PCH101; eBioscience) following manufacturer's protocol. The frequency of FoxP3 positive CD4+/CD25+ T cells under various culture conditions was enumerated by Flowjo analysis software. There were similar increases in CD25 expression in both the Tetanus- and Tregitope-stimulated samples indicating T cell activation by both peptides (FIG. 3; results shown for Tregitope-289). Expression of FoxP3 within the CD4+CD25+ subset, however, differed significantly depending on the stimulus used. Tetanus stimulation led to a 7% decrease in expression of FoxP3, whereas Tregitope stimulation led to a more than two-fold increase in expression, indicating Th and nTreg activation, respectively.

US10925942B2. Regulatory T cell epitopes, compositions and uses thereof (2021-02-23). EpiVax, Inc. [US]. Inventors: Anne De Groot [US], William Martin [US], Daniel S. Rivera [US].
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5

Multiparameter Immune Cell Profiling

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CD45 clone Hl30 (eBioscience 47–0459-42), CD3e clone OKT3 (eBioscience 46–0037-42), HLA-DR clone L243 (eBioscience 48–9952-42), CD56 clone CMSSB (eBioscience 46–0567-42), CD56 clone HCD65 (BioLegend 318304), CD19 clone H1B19 (eBioscience 46–0198-42 and 56–0199-42), CD14 clone 61D3 (Invitrogen Q10056; Cohort 1), CD14 clone M5E2 (BioLegend 301836; Cohort 2), CD16 clone 3G8 (BioLegend 302040), CD11c clone 3.9 (eBioscience 56–0116-42), CD85g clone 17G10.2 (eBioscience 12–5179-42), BDCA1 clone L161 (BioLegend 331516), BDCA3 clone AD5–14H12 (Miltenyi 130–090-513), CD4 clone RPA-T4 (BioLegend 300550), CD8 clone RPA-T8 (BioLegend 301040), CD25 clone BC96 (BioLegend 302612), CD25 clone 2A3 (eBioscience 17–0259-42), FoxP3 clone PCH101 (eBioscience 77–5776-40), FoxP3 clone 236A/E7 (eBioscience 25–4777-42), PD-1 clone EH-12 (BioLegend 329930), CTLA-4 clone BNI3 (BioLegend 369606), γδ TCR clone B1.1 (BioLegend 331212), Flt3L (Abcam ab9688).
Barry K.C., Hsu J., Broz M.L., Cueto F.J., Binnewies M., Combes A.J., Nelson A.E., Loo K., Kumar R., Rosenblum M.D., Alvarado M.D., Wolf D.M., Bogunovic D., Bhardwaj N., Daud A.I., Ha P.K., Ryan W.R., Pollack J.L., Samad B., Asthana S., Chan V, & Krummel M.F. (2018). A Natural Killer/Dendritic Cell Axis Defines Responsive Tumor Microenvironments in Melanoma. Nature medicine, 24(8), 1178-1191.
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6

Tregitope Induction of Regulatory T Cells

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Example 3

Human PBMCS were stimulated directly ex vivo for 4 days in the presence of tetanus toxin peptide TT830-844 alone, Tregitope-289 alone, phytohemagglutinin (a mitogenic positive control) alone, or no stimulus. 1×106 cells were stained with anti-CD4-FITC (clone RPA-T4; eBioscience) and anti-CD25-APC (clone BC96; eBioscience) antibodies for 30 minutes on ice in Flow Staining Buffer (eBioscience) and washed twice with buffer. Following cell-surface staining, cells were fixed and permeabilized (eBioscience) and stained intracellulary for Foxp3 (clone PCH101; eBioscience) following manufacturer's protocol. The frequency of FoxP3 positive CD4+/CD25+ T cells under various culture conditions was enumerated by Flowjo analysis software. There were similar increases in CD25 expression in both the Tetanus- and Tregitope-stimulated samples indicating T cell activation by both peptides (FIG. 3; results shown for Tregitope-289). Expression of FoxP3 within the CD4+CD25+ subset, however, differed significantly depending on the stimulus used. Tetanus stimulation led to a 7% decrease in expression of FoxP3, whereas Tregitope stimulation led to a more than two-fold increase in expression, indicating Th and nTreg activation, respectively.

US11045532B2. Regulatory T cell epitopes, compositions and uses thereof (2021-06-29). EpiVax, Inc. [US]. Inventors: Anne De Groot [US], William Martin [US], Daniel S. Rivera [US].
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7

Tregitope Induction of Regulatory T Cells

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Example 3

Human PBMCS were stimulated directly ex vivo for 4 days in the presence of tetanus toxin peptide TT830-844 alone, Tregitope-289 alone, phytohemagglutinin (a mitogenic positive control) alone, or no stimulus. 1×106 cells were stained with anti-CD4-FITC (clone RPA-T4; eBioscience) and anti-CD25-APC (clone BC96; eBioscience) antibodies for 30 minutes on ice in Flow Staining Buffer (eBioscience) and washed twice with buffer. Following cell-surface staining, cells were fixed and permeabilized (eBioscience) and stained intracellulary for Foxp3 (clone PCH101; eBioscience) following manufacturer's protocol. The frequency of FoxP3 positive CD4+/CD25+ T cells under various culture conditions was enumerated by Flowjo analysis software. There were similar increases in CD25 expression in both the Tetanus- and Tregitope-stimulated samples indicating T cell activation by both peptides (FIG. 3; results shown for Tregitope-289). Expression of FoxP3 within the CD4+CD25+ subset, however, differed significantly depending on the stimulus used. Tetanus stimulation led to a 7% decrease in expression of FoxP3, whereas Tregitope stimulation led to a more than two-fold increase in expression, indicating Th and nTreg activation, respectively.

US10925941B2. Regulatory T cell epitopes, compositions and uses thereof (2021-02-23). EpiVax, Inc. [US]. Inventors: Anne De Groot [US], William Martin [US], Daniel S. Rivera [US].
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8

Multiparametric Flow Cytometry Analysis of Treg and Th17 Cells

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0.5-1×106 PBMC/ml were stained using the FOXP3 staining kit (eBioscience, USA) according to manufacturer’s protocols with fluorochrome-labeled mAbs against human CD4 (clone RPA-T4), CD25 (clone 2A3), CD127 (clone hIL-7R-m21) IL-17 (clone N49-653), and CTLA-4 (Cytotoxic T-Lymphocyte Antigen 4, clone BN13) (all BD Biosciences, USA), GITR (glucocorticoid-induced TNFR-related protein, clone 110416, R&D, Germany) and FOXP3 (clone PCH101, eBioscience, USA), and analyzed by FACSCanto (BD Biosciences, USA).
IL-17 production was assessed after 3-hour stimulation of cells at 37°C in the presence of phorbol 12-myristate 13-acetate (PMA) (0.05 µg/mL), ionomycin (0.5 µg/mL), and brefeldin A (5 µg/mL) (Sigma-Aldrich, USA).
Vercoulen Y., Bellutti Enders F., Meerding J., Plantinga M., Elst E.F., Varsani H., van Schieveen C., Bakker M.H., Klein M., Scholman R.C., Spliet W., Ricotti V., Koenen H.J., de Weger R.A., Wedderburn L.R., van Royen-Kerkhof A, & Prakken B.J. (2014). Increased Presence of FOXP3+ Regulatory T Cells in Inflamed Muscle of Patients with Active Juvenile Dermatomyositis Compared to Peripheral Blood. PLoS ONE, 9(8), e105353.
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9

Multiparameter Flow Cytometry of PBMCs

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The phenotype of PBMCs cell subsets was evaluated by multiparameter flow cytometry. Cells were incubated in the dark for 20 min RT with the following panel of anti-human fluorescent labelled antibodies: CD3 (clone HIT3a, APC conjugated, BioLegend), CD4 (clone SK3, APC conjugated, eBiosciences), CD8 (clone SK1, APC conjugated, BD Biosciences), CD14 (clone 61D3, FITC conjugated, eBiosciences), CD25 (clone BC96, Alexa Fluor 488 conjugated, eBiosciences), CD69 (clone FN50, PE conjugated, eBiosciences), CD45 (clone L48, PerCyP conjugated, BD Biosciences), Foxp3 (clone PCH101, PE conjugatated, eBiosciences), IL-1β (clone CRM56, FITC conjugated, eBiosciences), IL-6 (clone MQ2-13A5, FITC conjugated, eBiosciences), IL-10 (clone JES3-9D7, Alexa Fluor 488 conjugated, eBiosciences), HLA-DR (clone L243, APC.Cy7 conjugated, BD Biosciences), CD33 (clone P67.6, PE conjugated, BD Biosciences), CD11b (clone D12, APC conjugated, BD Biosciences), CD14 (clone MɸP9, Brilliant Violet 421 conjugated, BD Biosciences) and Arginase-1 (ARG-1, FITC-conjugated, R&D Systems). Isotype controls were used for each experiment. After incubation, cells were again washed, resuspended in flow buffer and analyzed using FACSCalibur and FACSCanto II cytometers (BD, Biosciences). At least 5 × 104 events were collected, and the data was analyzed using Summit software (Summit Group Software).
Domenis R., Cesselli D., Toffoletto B., Bourkoula E., Caponnetto F., Manini I., Beltrami A.P., Ius T., Skrap M., Di Loreto C, & Gri G. (2017). Systemic T Cells Immunosuppression of Glioma Stem Cell-Derived Exosomes Is Mediated by Monocytic Myeloid-Derived Suppressor Cells. PLoS ONE, 12(1), e0169932.
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10

Multicolor Flow Cytometry of Immune Cells

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LL2 and MC38 cells were purchased from ATCC (Rockville, MD). Cells were cultured in vitro in RPMI-1640 media supplemented with 10% fetal bovine serum, 50 units/mL of penicillin/streptomycin, 2 mM of L-glutamine, 1 mM of sodium pyruvate, and 2 nM of non-essential amino acids, and grown at 37°C. For flow cytometric analyses, antibodies including CD11b (clone M1/70, Biolegend), Gr-1 (Clone RB6–8C5, Biolegend), Ly6C (clone HK1.4, Biolegend), Ly6G (Clone 1A8, Biolegend), RAE-1γ (clone CX1, Biolegend), CD4 (clone GK1.5), CD3ε (clone 145–2C11, BD Bioscience), NK1.1 (clone PK136, Biolegend), Foxp3 (clone PCH101, eBioscience), CD8 (53–6.7, Biolegend), IFNg (XMG1.2, Biolegend) and CD45 (30-F11, Biolegend) were used. Live cell gating was performed using Zombie Aqua Fixable Viability Kit (Biolegend). To detect NKG2D-Fc, we used the PE goat anti-mouse antibodies (Multiple Adsorption, BD Bioscience Cat No: 550589).
Feng P.H., Lam B., Tseng S.H., Kung Y.J., Farmer E., Cheng M.A, & Hung C.F. (2020). NKG2D-Fc fusion protein promotes antitumor immunity through the depletion of immunosuppressive cells. Cancer immunology, immunotherapy : CII, 69(10), 2147-2155.
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