Cd127 fitc

Manufactured by Miltenyi Biotec

Sourced in Germany

CD127-FITC is a fluorochrome-conjugated monoclonal antibody that binds to the CD127 (IL-7 receptor alpha) antigen. CD127 is expressed on the surface of various cell types, including T cells, B cells, and hematopoietic progenitor cells. The FITC (fluorescein isothiocyanate) fluorochrome allows for the detection and analysis of CD127-expressing cells using flow cytometry.

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5 protocols using cd127 fitc

Multiparametric Flow Cytometry Analysis

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The following mAbs were used for staining and flow cytometric measurements: CD3-VioGreen (BW264/56), CD4-allophycocyanin-Vio770 (VIT4), CD8-PE-Vio770 (BW135/80), CD14-peridinin chlorophyll protein (PerCP) (TÜK4), CD20-PerCP (LT20), CD27-FITC (M-T271), CD28-FITC (15E8), CD45RA-VioBlue (T6D11), CD45RO-FITC (UCHL1), CD62L-FITC (145/15), CD107a-PE (1D4B), CD127-FITC (MB15-18C9), CD137-allophycocyanin, CD137-PE (4B4-1), CD154-VioBlue (5C8), CD178-PE (NOK-1), CD197 (CCR7)-allophycocyanin, CD197 (CCR7)-PE (150503), CD279-allophycocyanin (PD1.3.1.3), anti-IL-2-PE-Vio770 (N7.48A), anti-IL-4-PE (7A3-3), anti-IL-17-FITC (CZ8-23G1), anti-IFN-γ-FITC, anti-IFN-γ-PE-Vio770 (45-15), anti-TNF-α-PE, and anti-TNF-α-VioBlue (cA2) (all Miltenyi Biotec).
Soluble biotinylated pHLA-A*0201 molecules loaded with WT1₃₇ (VLDFAPPGA), WT1₁₂₆ (RMFPNAPYL), WT1₁₈₇ (SLGEQQYSV), WT1₂₃₅ (CMTWNQMNL), or cytomegalovirus (CMV) pp65₄₉₅ (NLVPMVATV) were produced as described previously.14 (link) Tetramerization was achieved by binding to streptavidin-PE or streptavidin-allophycocyanin (both BioLegend, San Diego, CA). In each case, 2·5 × 10⁶ cells were stained with 1 µg/ml tetramer for 45 min at 4°C.
Data were acquired using a MACSQuant flow cytometer and analysed with MACSQuantify software (both Miltenyi Biotec).

Schmied S., Gostick E., Price D.A., Abken H., Assenmacher M, & Richter A. (2015). Analysis of the functional WT1-specific T-cell repertoire in healthy donors reveals a discrepancy between CD4+ and CD8+ memory formation. Immunology, 145(4), 558-569.

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Immune Cell Profiles in Pregnancy and Multiple Sclerosis

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CD4⁺ and CD8⁺ T lymphocytes, Treg, NKbright and M2 monocytes were evaluated in blood specimen of HC and MS patients at different trimester of pregnancy. In addition, the same populations were analyzed in decidual tissues. The analyses were performed by a biologist blinded to the clinical data.
Non-specific sites of 3 × 10⁶ cells were blocked with rabbit immunoglobulins G (IgG, Sigma-Aldrich), and cells were then incubated with fluorochrome-conjugated monoclonal Ab (mAb) and isotype–matched negative controls for 10 min at 4°C. The following anti-human mAbs were used: CD163 Phycoerythrin (PE) and CD14 Fluorescein isothiocyanate (FITC) for M2 monocytes, CD3 allophycocyanin (APC)-Vio770, CD16 FITC and CD56 APC for NK cells, CD3 APC-Vio770, CD4 PE-Vio770 and CD8 FITC for T cells, CD4 PE-Vio770, CD25 APC, and CD127 FITC for Treg (Miltenyi Biotec, Bergisch Gladbach, Germany). Living cells identified by propidium iodide (Sigma-Aldrich) exclusion were gated according to their light-scatter properties to exclude cell debris. Samples were analyzed using a CyAn ADP, running Summit 4.3 software (Beckman Coulter, Brea, CA, USA). The gating strategy is shown in the Figure 1.

Spadaro M., Martire S., Marozio L., Mastromauro D., Montanari E., Perga S., Montarolo F., Brescia F., Balbo A., Botta G., Benedetto C, & Bertolotto A. (2019). Immunomodulatory Effect of Pregnancy on Leukocyte Populations in Patients With Multiple Sclerosis: A Comparison of Peripheral Blood and Decidual Placental Tissue. Frontiers in Immunology, 10, 1935.

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Western Blot Antibody Compendium

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The following antibodies used for western blot analyses were obtained from Cell Signaling Technology (unless otherwise indicated): phospho-AKT^S473 (#9271), phospho-ERK1/2 (#4370), phospho-JAK1 (#3331), phospho-JAK2 (#3771), phospho-MEK1/2 (#9154), phospho-mTOR (#2971), phospho-p70S6Kinase (#9204), phospho-STAT1 (#9167), phospho-STAT3 (#9145), phospho-STAT5 (#9351), phospho-TYK2 (#9321), DYKDDDDK (#2368), CD127 (anti-IL7Ra; R&D Systems, Minneapolis, MN, USA; #MAB306), RAS (Merck Millipore; #05-516) and β-actin (Sigma-Aldrich; #2547). The following antibodies used for flow cytometry were obtained from Miltenyi Biotec: CD127-FITC (#130-094-888) and CD271-APC (#130-091-884).

Canté-Barrett K., Spijkers-Hagelstein J.A., Buijs-Gladdines J.G., Uitdehaag J.C., Smits W.K., van der Zwet J., Buijsman R.C., Zaman G.J., Pieters R, & Meijerink J.P. (2016). MEK and PI3K-AKT inhibitors synergistically block activated IL7 receptor signaling in T-cell acute lymphoblastic leukemia. Leukemia, 30(9), 1832-1843.

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Regulatory T-Cell Levels in Autoimmune Diseases

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Regulatory T-cell levels were evaluated in 29 HTE, 17 HTI, 22 MS, 12 MS + HT, and 33 HC. After gentle thawing at 37°C, PBMCs were immediately added to 5-mL RPMI 1640 (Thermo Fisher Scientific), supplemented with 10% FBS (Thermo Fisher Scientific) and centrifuged to remove DMSO (Sigma-Aldrich). Samples were resuspended in RPMI 1640 medium supplemented with 10% heat-inactivated FBS and counted for flow-cytometry experiments.
For Treg evaluation, PBMCs were incubated for 5 min at 4°C with rabbit immunoglobulins G (IgG, Sigma-Aldrich) to block non-specific sites and then for 10 min at 4°C with fluorochrome-conjugated monoclonal Ab (mAb) or isotype-matched negative controls. For Treg determination the following antihuman mAbs were used: CD4 PE-Vio770, CD25 APC, and CD127 FITC (Miltenyi Biotec, Bergisch Gladbach, Germany). Living cells identified by propidium iodide (Sigma-Aldrich) exclusion were gated according to their light-scatter properties to exclude cell debris. Samples were analyzed using a CyAn ADP, running Summit 4.3 software (Beckman Coulter, Brea, CA, USA).

Perga S., Martire S., Montarolo F., Giordani I., Spadaro M., Bono G., Corvisieri S., Messuti I., Panzica G., Orlandi F, & Bertolotto A. (2018). The Footprints of Poly-Autoimmunity: Evidence for Common Biological Factors Involved in Multiple Sclerosis and Hashimoto’s Thyroiditis. Frontiers in Immunology, 9, 311.

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Flow Cytometry Analysis of Cell Samples

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Cell sample analysis by flow cytometry was performed at multiple times before, during and after the CliniMACSprocess to monitor the quality of the cell product. The expression of CD4, CD8, CD14, CD15, CD20, CD25, CD45, CD127, and FOXP3 was analyzed. Red blood cells were eliminated from the cell samples by the use of red blood cell lysis solution (Miltenyi Biotec) prior to antibody staining. Cell viability was analyzed using Via-Probe 7-Amino-actinomycin D (7-AAD) solution (Miltenyi Biotech) (Supporting Information Fig. S2). Cells were prepared for intracellular staining using FoxP3 Staining Buffer Set (eBioscience, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. The following antibodies were used for analysis: CD8-APC, CD14-PE, CD15-PE, CD20-FITC, CD45-VioBlue, CD127-FITC, Treg-Detection-kit, all from Miltenyi Biotec; CD4-BV421 and CD25-PE from BioLegend, San Diego, CA, USA; FoxP3-Alexa Fluor 647 from BD Biosciences. All flow cytometric analyses were performed on a FACSVerse TM (BD Biosciences), and the data were analyzed by FLOWJO version 10.7.0 (FlowJo LLC, Ashland, OR, USA).

Bergström M., Yao M., Müller M., Korsgren O., von Zur-Mühlen B, & Lundgren T. (2021). Autologous regulatory T cells in clinical intraportal allogenic pancreatic islet transplantation. Transplant international : official journal of the European Society for Organ Transplantation, 34(12).

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