PBMC were labeled with PE anti-CD127 (#557938, BD Biosciences), PE-Cy7 anti-CD4 (#557852, BD Biosciences), and APC anti-CD25 (#561399, BD Biosciences) and sorted using a BD Biosciences FACSAria IIIu into CD4+CD127low/-CD25+ Treg, nonTreg lymphocytes, and monocytes. The sorted cell populations were recombined to preserve original proportions, except that medium was substituted for Treg in the Treg-depleted group. Cells rested overnight before labeling and stimulation with sBc as described above.
Anti cd127 pe
Manufactured by BD
Sourced in United States
Anti-CD127-PE is a fluorescently-labeled monoclonal antibody that binds to the CD127 (IL-7 receptor alpha) cell surface antigen. It is used for the identification and enumeration of CD127-expressing cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd25 apc, by BD (6 mentions)
Anti cd8 fitc, by BD (5 mentions)
Anti cd25 fitc, by BD (4 mentions)
Anti cd4 fitc, by BD (3 mentions)
Anti cd4 apc ef780, by BD (3 mentions)
Anti cd122 pe, by BD (3 mentions)
Mccoy s 5a medium, by Thermo Fisher Scientific (2 mentions)
Anti cd4 pe cy7, by BD (2 mentions)
Skov3, by American Type Culture Collection (2 mentions)
Anti cd4 percp, by BD (2 mentions)
Anti cd45ra fitc, by BD (2 mentions)
Ficoll paque plus, by GE Healthcare (2 mentions)
Anti cd27 fitc, by BD (2 mentions)
Anti cd3 apc cy7, by BD (2 mentions)
Cyan adp flow cytometer, by Beckman Coulter (2 mentions)
Anti cd3 percp, by BD (2 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Anti cd44 fitc, by BD (2 mentions)
Anti cd62l pe, by BD (2 mentions)
Anti cd38 pe, by BD (2 mentions)
18 protocols using anti cd127 pe
1
FACS Sorting of Treg Subsets
2
Sorting of CD4+ Treg and Teff cells
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CD4+ Tregs and Teffs were sorted from PBMCs by fluorescence-activated cell sorting (FACS), following staining with FITC-anti-CD4, APC-anti-CD25, PE-anti-CD127 (all from BD Biosciences, San Jose, CA, USA), using a FACS Aria II cell sorter; the purity of the sorted cells was >95%.
The human OC cell line SKOV3 was purchased from ATCC (American Type Culture Collection, Manassas, VA, USA), and cultured in McCoy’s 5 A medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS at 37 °C in 5% CO2.
The human OC cell line SKOV3 was purchased from ATCC (American Type Culture Collection, Manassas, VA, USA), and cultured in McCoy’s 5 A medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS at 37 °C in 5% CO2.
3
Immunophenotyping of Regulatory T Cells
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Freshly isolated PBMCs (1×10 6 ) were washed and incubated with uorophore-conjugated monoclonal anti-bodies including Anti-CD4-FITC, Anti-CD25-APC, Anti-CD127-BV421 for 30 min at room temperature in the dark. For intracellular staining, PBMCs (1×10 6 ) were washed, xed, permeabilized, and stained with Anti-mTOR-PE according to the manufacturer's protocol. For intranuclear transcription factor detection, cells (1×10 6 ) were washed, xed, permeabilized, and stained with 4E-BP1 Rabbit mAb and Anti-rabbit IgG (H + L), F(ab')2 Fragment (PE Conjugate) according to the manufacturer's protocol. Fluorescence data were collected on a FACS Aria II (BD Biosciences, San Jose, CA, USA) and analyzed with FlowJo software (Tree Star, Ashland, OR, US).
Cell sorting and cell line culture CD4 + Tregs and Teffs were sorted from PBMCs by FACS, following staining with FITC-anti-CD4, APC-anti-CD25, PE-anti-CD127 (all from BD Biosciences, San Jose, CA, USA), using a FACS Aria II cell sorter; the purity of the sorted cells was > 95%.
The human OC cell line SKOV3 was purchased from ATCC (American Type Culture Collection, Manassas,VA, USA), and cultured in McCoy's 5A medium (Invi-trogen, Carlsbad, CA, USA) supplemented with 10% FBS at 37°C in 5% CO 2 .
Cell sorting and cell line culture CD4 + Tregs and Teffs were sorted from PBMCs by FACS, following staining with FITC-anti-CD4, APC-anti-CD25, PE-anti-CD127 (all from BD Biosciences, San Jose, CA, USA), using a FACS Aria II cell sorter; the purity of the sorted cells was > 95%.
The human OC cell line SKOV3 was purchased from ATCC (American Type Culture Collection, Manassas,VA, USA), and cultured in McCoy's 5A medium (Invi-trogen, Carlsbad, CA, USA) supplemented with 10% FBS at 37°C in 5% CO 2 .
4
Phenotypic Analysis of Regulatory T Cells
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Human peripheral blood mononuclear cells (PBMCs) were isolated from freshly drawn venous blood and BM mononuclear cells (BMMCs) were obtained from BM in both of OA and RA patients by Ficoll-Paque PLUS (GE Healthcare Biosciences AB, Uppsala, Sweden) density centrifugation. To examine the phenotype of regulatory T cells (Tregs), the following antibodies were used: anti-CD4-PerCP, anti-CD25-FITC, anti-CD127-PE, anti-CD45RA-FITC, anti-CD45RO-PE and anti-CD184 (CXCR4)-PE (all from BD Biosciences, San Jose, CA, USA). Next, cells were fixed and permeabilized using the Foxp3 Staining Buffer Set followed by intracellular staining with anti-Foxp3-APC antibody (PCH101; eBioscience, San Diego, CA, USA). Cells were acquired using FACSCalibur (BD Biosciences), and the results were analyzed using CellQuest (BD Biosciences) software. The gating strategy was based on the identification of lymphocytes according to FSC and SSC signal distribution. Then, CD4+ lymphocytes were gated, and next, appropriate cell populations were identified. Gates were settled according to the isotype control or fluorescence minus one (FMO) for the desired marker. The dot plots shown are representative for at least six experiments done on different patients. The described gating strategy is shown in Figure 2b and was used though all the analysis of the cell phenotype presented in this paper.
5
Comprehensive Immunophenotyping of T Cells
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Cells were stained with 7-AAD viability dye, anti-CD3 eFluor450, anti-CD4 PE-eFluor 647 (eBioscience), anti-CD4 electron coupled dye (Beckmann Coulter), anti-CD4 fluorescein isothiocyanate (FITC), anti-CD3 PE, anti-CD3 APC-Cy7, anti-CD8 PE, anti-CD8 APC-Cy7, anti-CD25 PECy7, anti-CD127 PE, and anti-CD27 FITC (BD), anti-CD45RA FITC, FOXP3 Alexa Fluor 647 (BioLegend) specific antibodies. FOXP3 intracellular staining was performed using FOXP3 staining buffers (eBioscience). Data were acquired using a fluorescence activated cell sorting (FACS)CantoII and analysed using FACSDiva software (BD) and Flojo software (Treestar).
6
Mouse Peripheral Blood Immune Profiling
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Mouse peripheral blood was obtained by retro-orbital venipuncture, collected in sodium citrate solution, red blood cells lysed and the remaining cells were washed in staining buffer (2% BSA, 0.02% azide in PBS (PAB)) as previously described (Bustamante et al 2008, 2014, 2020). Whole blood was incubated with a major histocompatibility complex I (MHC I) tetramer containing the specific TSKB20 peptide (ANYKFTLV/Kb) labeled with BV421 (Tetramer Core Facility at Emory University, Atlanta, GA) and the following labeled antibodies: anti-CD8 FITC, anti-CD4 APC EF780, anti-CD127 PE (BD Bioscience, San Jose, CA). For whole blood, we lysed red blood cells in a hypotonic ammonium chloride solution after washing twice in PAB. We stained cells for 45 min at 4°C in the dark, washed them twice in PAB and fixed them in 2% formaldehyde. At least 500,000 cells were acquired using a CyAn ADP flow cytometer (Beckman Coulter, Hialeah, Florida) and analyzed with FlowJo software v10.6.1 (Treestar, Inc., Ashland, OR).
7
Immune Cell Profiling from Peripheral Blood
8
Isolation and Characterization of Human T Regulatory Lymphocytes
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Human T Regulatory Lymphocyte Isolation Set, anti-CD127-PE, anti-CD45RO-FITC, anti-CD25-APC, PE and APC conjugated streptavidin, and human recombinant IL-2 protein were purchased from BD Biosciences (San Jose, CA, USA). All flow cytometry and flow sorting experiments were done on FACSCalibur flow cytometer and data were analysed with CellQuest software (BD Biosciences). RPMI-1640 medium, anti-CD3/CD28 coated bead, and TRIzol reagent were from Life Technologies (Carlsbad, CA, USA). Foetal bovine serum (FBS) was obtained from HyClone Laboratories, Inc. (South Logan, Utah, USA). Anti-CD4-PerCP, biotinylated anti-IL-1R1, anti-IL-1R2, and Human sIL-1R2 Quantikine ELISA Kit were from R&D Systems (Minneapolis, MN, USA), and anti-GARP-PE (LRRC32) was from ENZO Life Sciences (Farmingdale, NY, USA). Antibiotic/Antimycotic Solution, L-glutamine, MEM's Vitamin Solution, and sodium-azide were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Ficoll Paque was acquired from GE Healthcare Biosciences (Uppsala, Sweden).
9
Comprehensive Immune Cell Profiling
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Splenocytes were treated with ACK buffer for red cell lysis and washed with RPMI with 10% FBS. The spleen, heart, lymph node, and liver cells were stained with H2Kk-TEWETGQI multimer for 10 min at RT. The cell surface was stained for 30 min at 4°C. The following antibodies were used for surface staining: anti-CD3 APCcy7 (clone 145-2C11, BD), anti-CD8 PERCP or anti-CD8 PACIFIC BLUE (clone 53-6.7, BD), anti-CD11a FITC (clone 2D7, BD), anti-CD11c APCcy7 (clone HL3, BD), anti-CD44 FITC (clone IM7, BD), anti-CD62L PE (clone MEL-14, BD), anti-CXCR3 PERCP/Cy5.5 (clone 173, BioLegend), anti-CD27 FITC (clone LG3A10, BD), anti-CD4 PEcy7 (clone RM4-5, BD), anti-KLRG1 FITC (clone 2F1, eBioscience), anti-CD49d PEcy7 (clone R1-2, BD), anti-CD69 PERCP (clone H1.2F3, BD), anti-CD43 PEcy7 (1B11, BioLegend), anti-CD95 PEcy7 (clone JO2, BD), anti-CD25 FITC (clone LG3A10, BD), anti-CD127 PE (clone SB/199, BD), anti-CD122 FITC (clone TM-β1, BD), anti-CD38 PE (clone 90, BD), anti-β7 PERCP (clone FIB27, BioLegend), anti-CD31 FITC (clone MEC 13.3, BD), anti-CD272 PE (clone 8F4, eBioscience), anti-PD-1 FITC (clone J43, eBioscience), anti-CTLA-4 PE (clone UC10-4B9, eBioscience), and anti-CCR7 PE (clone 4B12, BD). At least 500,000 cells were acquired on a BD FACS Canto II flow cytometer and analyzed with FlowJo 8.7.
10
Multiparametric Flow Cytometry Analysis
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Freshly isolated responders or the generated cells were analyzed with flow cytometry on days 0, 7, and 14. For the analysis of lymphocyte and monocyte lineage markers, the following conjugated antibodies (Abs) were used: anti-CD4-PerCP Cy5.5, anti-CD8-APC H7, anti-CD3-HV450, and anti-CD19-HV500 (all from BD Biosciences, San Jose, USA); and anti-CD16-PE, anti-CD56-PE, anti-CD14-PC7, and anti-CD45-APC AF700 (all from Beckman Coulter, Marseille, France). CD subsets were determined using the Lineage Cocktail 1 (lin 1; FITC-conjugated Abs against CD3, CD14, CD16, CD20, and CD56; all from BD Biosciences), anti-CD123-PECF594, anti-CD11c-PC7 (both from BD Biosciences), and anti-HLA DR-APC (Beckman Coulter). Tregs were identified using anti-CD127-PE, anti-CD4-PerCP Cy5.5 (BD Biosciences), anti-CD25 PC7 (Biolegend, San Diego, USA), and anti-FOXP3-AF647, clone 236A/E7 (BD Biosciences). The staining procedure, including fixation and permeabilization, was performed according to the manufacturer’s instructions (eBiosciences, San Diego, CA, USA). To prevent non-specific binding, mouse immunoglobulin G1 was added prior to incubation with the FOXP3 antibody. The data were acquired with a Navios flow cytometry instrument and analyzed using Kaluza software (Beckman Coulter, Brea, USA).
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