Isolated PBMC or cultured cells were analyzed for expression of surface markers using multi-color flow cytometry. Cell aliquots were treated with anti-human Fc mAb for 20 minutes and stained for 30 minutes with selected combinations of fluorochrome-conjugated antibodies: anti-human CD4-Alexa-PE 700 (Invitrogen, Carlsbad, CA), CD8-APC/Cy7 (BD Pharmingen, San Jose, CA), CD28-PE (BD Pharmingen, San Jose, CA), CD45RO-PerCP/Cy5.5 (BioLegend, San Diego, CA), CD25-PE/Cy7 (BioLegend, San Diego, CA), CD127-APC (BioLegend, San Diego, CA), ICOS-PerCP/Cy5.5 (BioLegend, San Diego, CA), IL15Rα (CD215)-PerCP (R&D systems, Minneapolis, MN), and CD107a-PE/Cy5 (BD Pharmingen, San Jose, CA). The cells were analyzed on a BD LSR II (BD Biosciences, San Jose, CA). Flow cytometry data was analyzed by FlowJo software version 9.4.5 (Tree Star, Inc. Ashland, OR).
Cd127 apc
Manufactured by BioLegend
Sourced in United States
CD127-APC is a fluorochrome-conjugated antibody used for the detection and quantification of the CD127 (IL-7 receptor alpha chain) molecule on the surface of cells. CD127 is a transmembrane protein that serves as the receptor for the cytokine interleukin-7 and is expressed on various lymphocyte subsets.
Automatically generated - may contain errors
Lab products found in correlation
Cd25 pe, by BioLegend (4 mentions)
Cd25 pe cy7, by BioLegend (3 mentions)
Cd3 fitc, by BioLegend (3 mentions)
Cd3 fitc, by BD (3 mentions)
Cd4 percp cy5, by BioLegend (2 mentions)
Pd1 apc, by BioLegend (2 mentions)
Tim 3 pe, by BioLegend (2 mentions)
Cd25 pe, by Thermo Fisher Scientific (2 mentions)
Cd4 fitc, by BioLegend (2 mentions)
Foxp3 pe, by Thermo Fisher Scientific (2 mentions)
Facscanto 2, by BD (2 mentions)
Percp cy5.5 cd4, by BD (2 mentions)
Cd45ra pe cy7, by BD (2 mentions)
Foxp3 pe, by BD (2 mentions)
Live dead fixable stain dye, by Thermo Fisher Scientific (2 mentions)
Cd25 bv421, by BD (2 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (2 mentions)
Ionomycin, by Thermo Fisher Scientific (2 mentions)
Cd11b fitc, by BioLegend (2 mentions)
Monensin, by Thermo Fisher Scientific (2 mentions)
16 protocols using cd127 apc
1
Multi-Color Flow Cytometry for Immune Cell Profiling
2
Induction and Phenotyping of Tregs
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The same procedure was followed as described in DC‐T cell co‐culture procedure and maintained for up to 5 days for induction of T regulatory cells (Tregs). On the 5th day, cells were harvested and stained for Treg‐specific markers; anti‐mouse CD4‐FITC (# 11‐0042‐82), CD25‐PE‐Cy7 (# 25‐0251‐82), and FOXP3‐PE (# 12–5773‐82) all from eBioscience (San Diego, CA); and CD127‐APC (# 158205) Biolegend (San Diego, CA) fluorochrome‐tagged monoclonal antibodies.
3
Comprehensive Immune Phenotyping Protocol
4
Isolation and Characterization of Human Regulatory T Cells
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Human PBMC were purified from healthy blood donors by density gradient centrifugation at 2000 rpm for 20 min with Biocoll Separating Solution (Biochrom, Darmstadt, Germany). Erythrolysis was performed after which the cell suspension was passed through a 0.45 mm filter (BD, Franklin Lakes, NJ, USA). Human Treg (CD4 + CD25 + CD127dim/- cells) were obtained from freshly isolated PBMC by magnetic-activated cell sorting (MACS) using a CD4 + CD25 + CD127dim/- Regulatory T-cell Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer`s protocol in a two-step isolation. Firstly, non-CD4 + and CD127high cells were removed by negative magnetic selection and, secondly, CD25 + cells were collected using CD25 positive selection magnetic beads. Purity of the isolation was confirmed by flow cytometry and found to be around at 75% SD (Supplement 2 ). Next, the cells were labelled with CD4-PE, CD127-APC, CD25-FITC, CD19-PacBlue and CD3-APC (all BioLegend, San Diego, CA, USA). Intracellular staining for FoxP3 was performed using a PacBlue anti-human FoxP3 Antibody and FoxP3 Fix/Perm Buffer Set (BioLegend, San Diego, CA, USA) following the manufacturer’s protocol. To check the quality of the isolation, a Zombie NIR Fixable Viability Kit (BioLegend, San Diego, CA, USA) was added once after which a viability over 90% was found in each isolation fraction.
5
Multiparametric Phenotyping of CD8+ T Cells
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Gp33‐41 peptide (KAVYNFATM) and gp61‐80 (GLNGPDIYKGVYQFKSVEFD) peptides were purchased from Mimotopes. PE‐conjugated gp33‐MHC class I tetramer (H‐2Db/gp33‐41) was kindly provided by the NIH tetramer core facility. For exclusion of dead cells, efluor780 (eBioscience) or ZombieAqua (BioLegend) was used. Antibodies used were as follows: PD‐1‐FITC (J43; Thermo Fisher Scientific), HVEM‐APC (LH1; eBioscience), CD19‐APC‐Cy7 (6D5; BioLegend), CD4‐PE (RM4‐5; BD Bioscience), CD4‐PerCP‐Cy5.5 (RM4‐5; BioLegend), CD8a‐FITC (53‐6.7, eBioscience), CD8a‐PE‐Cy7 (53‐6.7; BioLegend), CD8a‐APC‐Cy7 (53‐6.7; BioLegend), CD8a‐PE (53‐6.7; BD Bioscience), IFNγ‐APC (XMG1.2; BioLegend), CD127‐APC (SB/199; BioLegend), CD25‐PE (PC61; eBioscience), Ki‐67‐PE (16A8; BioLegend), KLRG‐1‐PerCP‐eFluor710 (2F1; eBioscience), CD62L‐BV421 (MEL‐14; BioLegend), CD45.1‐PerCP‐Cy5.5 (A20; BioLegend), TNFα‐PE (MP6‐XT22; BD Bioscience), and TNFα‐FITC (MP6‐XT22; BioLegend).
6
Immunophenotyping of Activated T-Cell Subsets
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PBMCs underwent incubation with anti-human CD4-FITC (Clone A161A1), CD25-PE/CY7 (Clone BC96), CD127-APC (Clone A019D5), and CXCR5-PE (Clone J252D4) (all from BioLegend, USA), at 4°C in the dark for 30 min; the corresponding isotype controls were utilized for specificity verification. After surface staining, the FACSARIAIII flow cytometer (BD Bioscience, USA) was used for detection of cells. Then, flow cytometry data were analyzed by FlowJo 7.6.1 (Treestar Inc., USA) (Supplementary Figure S1).
7
Multicolor Flow Cytometry Analysis
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For analysis of cell surface markers, cells were stained in PBS containing 2% heat-inactivated FBS with 2 mM EDTA supplementation, unless noted otherwise. Antibodies used were as follows: CD4-PerCP/Cy5.5, CD8α-APC, and CD127-APC (BioLegend); CD4-PE and CD44-PE (BD); CD62L-FITC, CD25-PE, CD45.2-FITC, and CD45.1-PE (eBioscience). Foxp3-APC and eFluor780 (eBioscience), Annexin V-FITC, and 7-AAD (BD) were stained according to the manufacturer’s instructions. Cells were analyzed with FACSCalibur or FACSCanto II flow cytometry (BD) using FlowJo software (Tree Star).
8
Detailed Immune Cell Phenotyping
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Cells were stained with the fluorochrome-conjugated monocolonal antibodies against CD4-FITC (BD Biosciences, 555346), CD25-PE (Biolegend, 302606), CD127-APC (Biolegend, 351315), HLA-ABC-PE (Biolegend, 311406), HLA-DR-FITC (Biolegend, 307604), isotype control-FITC (eBioscience, 11-4714), isotype control-PE (eBioscience, 12-4714), and isotype control-APC (BD Biosciences, 550854). After washing with phosphate-buffered saline (PBS), the cells were subsequently detected by flow cytometry using FC500MCL (Beckman Coulter) and data were calculated using FlowJo Software (Tree Star).
9
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).
10
Immunotherapy for Prostate Cancer in Mice
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Six-week-old FVB/NJ mice were implanted with 1×106 MyC-CaP cells administered subcutaneously. Only male mice were used for these studies, given that the tumor of origin is male specific. Each mouse was then immunized intradermally with 100 μg DNA vaccine (pTVG-AR), or vector control (pTVG-4) weekly, beginning 1 day after tumor implantation. TLR agonists were coadministered with the vaccine intradermally at the following doses: TLR3 (Poly(I:C) HMW, 100 µg/mouse), TLR9 (ODN 1826, 50 µg/mouse). Mice were further treated with immune checkpoint blockade, αPD-1, αLAG-3, αCTLA-4, or IgG control the day following each immunization. Tumor volumes were measured as described above. In a parallel study, tumors were collected after treatment and digested in collagenase, DNAse I, and protease inhibitors for 2 hours at 37°C, passed through a 100 mm mesh screen, and analyzed by flow cytometry with the following antibodies: CD3-FITC (BD 555274), CD4-BUV395 (BD 563790), CD8-BV785 (BD 563332), CD25-PE-CF594 (BD 562694), CD44-PE-Cy7 (BD 561283), CD45-BV510 (BD 563891), CD62L-BV605 (BD 563252), CD127-APC (Biolegend 121122), KLRG1-BV711 (BD 564014), FOXP3-PE (Thermo Fisher 12-5773-82), 4-1BB-PerCP-eF710 (Lifetech 46-1371-82), and Live/Dead Ghost dye 780 (Tonbo, San Diego, CA 13-0865-T100).
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