TregFlowEx® Kit

ABIN3072918 产品详细信息, 供应商: Log in to see
应用范围
Flow Cytometry (FACS)
选项
Supplier
Log in to see
Supplier Product No.
Log in to see
原理 The TregFlowEx® Kit is designed for the detection of regulatory T cells (CD4+CD25+FOXP3+ cells) in human peripheral blood or human umbilical cord blood using flow cytometry.
品牌 TregFlowEx®
样品类型 Blood
组件
  • Fix and Lysing Solution
    1 vial containing 10 ml of 10x concentrated reagent.
  • Permeabilizing Solution
    1 vial containing 25 ml of reagent, ready-to-use.
  • Blocking Buffer
    1 vial containing 2.5 ml of reagent, ready-to-use.
  • CD4 FITC/CD25 PE
    1 vial containing 0.50 ml of cell surface staining reagent, a mixture of mouse monoclonal antibody against CD4, FITC labelled (clone MEM-241, isotype IgG1) and antibody against CD25, RPE labelled (clone MEM-181, isotype IgG1.
  • FOXP3 APC
    1 vial containing 0.25 ml of monoclonal antibody against FOXP3, APC labelled (clone 3G3, isotype IgG1).
试剂未包括
  • Suitable 5 mL test tubes for blood staining (e.g. 12 x 75 mm)
  • PBS buffer
  • Automatic pipettes with disposable tips
  • Vortex mixer
  • Refrigerated centrifuge with rotor suitable for test tubes
  • Flow cytometer - blue laser excitation at 488 nm, red laser excitation at 633 nm and proper emission filters
  • 1 % formaldehyde solution in PBS
  • Liquid waste container with appropriate disinfectant
  • Pasteur pipettes and vacuum source for aspiration of supernatants from test tubes
背景 Regulatory T-cells (Treg cells) are a subset of lymphocytes that have immune suppressive properties and play a critical role in the maintenance of self-tolerance. Their regulatory function leads to protection of tissues from collateral damage triggered by immune responses against microbes and allergens, they facilitate maternal tolerance to allogeneic fetus during pregnancy and maintain homeostasis with commensal microbiota [1]. Tregs are usually characterized as CD4+ CD25high T-cells expressing forkhead box transcription factor (FOXP3). Such phenotype is found in 5-12 % of human peripheral blood CD4+ T-cells [2]. Tregs can be further distinguished into the so-called "natural" Tregs originating in thymus (tTreg cells) and the peripherally induced Tregs (pTreg cells) [3]. The discrimination between tTregs and pTregs is still disputable and several markers were proposed to be the tTreg unique features: the expression of Helios transcription factor and the expression of Neutropilin-1, a receptor for members of the vascular endothelial growth factor family [4]. [1] Sakaguchi S, Miyara M, Constantino CM, Hafler DA. FOXP3+ regulatory T cells in the human immune system. Nat Rev Immunol (2010) 10:490-500. [2] Churlaud G, Pitoiset F, Jebbawi F, Lorenzon R, Bellier B, Rosenzwajg M, Klatzmann D. Human and Mouse CD8(+)CD25(+)FOXP3(+) Regulatory T Cells at Steady State and during Interleukin-2 Therapy. Front Immunol. 2015 Apr 15,6:171. [3] Abbas AK, Benoist C, Bluestone JA, Campbell DJ, Ghosh S, Hori S, Jiang S, Kuchroo VK, Mathis D, Roncarolo MG, Rudensky A, Sakaguchi S, Shevach EM, Vignali DA, Ziegler SF. Regulatory T cells: recommendations to simplify the nomenclature. Nat Immunol. 2013 Apr,14(4):307-8. [4] Lin X, Chen M, Liu Y, Guo Z, He X, Brand D, Zheng SG. Advances in distinguishing natural from induced Foxp3(+) regulatory T cells. Int J Clin Exp Pathol. 2013,6(2):116-23.
样本量 100 μL
实验流程 This test is based on combined cell surface and intracellular detection of CD4, CD25 and FOXP3 proteins that are characteristic for the phenotype of regulatory T cells. Blood samples are initially stained with a mixture of anti-CD4 (fluorescein labelled) and anti-CD25 (R-phycoerythrin labelled) monoclonal antibodies which specifically bind to the antigenic determinants expressed on the surface of leukocytes. Specific staining of blood cells is followed by lysis of red blood cells. Afterwards, leukocytes are permeabilized and stained with monoclonal antibody against FOXP3 protein (allophycocyanine labelled). The fluorochromes attached to the monoclonal antibodies are excited by appropriate laser beam and emission of light from each fluorochrome on individual cells is collected and analyzed by a flow cytometer.
试剂准备

Reagents preparation

  • Fix and Lysing Solution
    • Fix and Lysing Solution is provided as 10x concentrated solution that needs to be diluted with deionized water. Dilute only the necessary amount of the concentrate (1 mL of the diluted solution is needed per reaction) and store the diluted solution at 2-8 °C. The diluted solution is stable for 1 month.
  • Permeabilization Solution
    • Bring Permeabilization Solution to room temperature. Check for SDS precipitation and if necessary, dissolve the SDS by warming the vial to 37 °C. Afterwards store the Permeabilizing Solution at room temperature (15-25 °C). Storage at room temperature does not affect its shelf life.
  • PBS buffer
    • Prepare PBS buffer according to recipe below (other recipes may apply).
      • Dissolve:
      • 8.0 g of Sodium Chloride (NaCl)
      • 0.2 g of Potassium Chloride (KCl)
      • 2.0 g of Potassium Phosphate monobasic (KH2PO4)
      • 1.42 g of Sodium Phosphate dibasic dihydrate (Na2HPO4•2H2O) in 800 mL of deionized H2O.
    • Adjust the pH to 7.4 with HCl.
    • Add deionized H2O to 1 liter.
    • Sterilize by autoclaving for 20 minutes at 15 psi or by filter sterilization.
    • Store at 2 - 8 °C.
  • 1 % formaldehyde in PBS
    • Prepare 1 % formaldehyde in PBS buffer by mixing 1 part of methanol stabilized 37 % formaldehyde solution and 36 parts of PBS buffer. Store at 2 - 8 °C. The solution is stable for 1 month.
    • Warning: The concentrated formaldehyde solution is classified as acutely toxic substance. Refer to the safety precautions provided by your formaldehyde supplier.

样品制备

Human blood or human umbilical cord blood

Blood samples should be collected into tubes containing anticoagulant, e.g. EDTA, citrate or heparin. It is recommended to use freshly withdrawn samples. Cell viability decreases in time after collection and may directly influence results due to possible loss of specific cell subsets. A non-specific binding of monoclonal antibody reagents to non-viable cells occurs and may negatively influence test results. Store the blood samples at room temperature.

实验流程

Surface antigen staining

  1. Add 10 μL of CD4 FITC/CD25 PE antibody cocktail per tube.
  2. Add 100 μL of human blood.
  3. Mix well and incubate for 10 minutes in the dark in the refrigerator (2-8 °C).
  4. Wash cells by adding 2 mL of cold PBS buffer and centrifuge at 300 g for 5 minutes at 4 °C. Aspirate supernatant completely.
  5. Proceed immediately to intracellular antigen staining.

Intracellular antigen staining
  1. Add 1 mL of cold Fix and Lysing Solution (the 10x diluted solution).
  2. Mix well and incubate for 10 minutes in the dark in the refrigerator (2-8 °C).
  3. Add 0.5 mL of room temperature Permeabilizing Solution.
  4. Mix well and incubate for 10 minutes in the dark in the refrigerator (2-8 °C).
  5. Centrifuge the cells for 5 minutes at 400 g at 4 °C.
    (Do not add wash buffer to the Fix/Perm mixture)
  6. Remove (decant) supernatant.
  7. Add 50 μL of cold Blocking Buffer
  8. Add 50 μL of cold PBS buffer
  9. Mix well and incubate for 5 minutes in the dark in the refrigerator (2-8 °C).
  10. Add 5 μL of FoxP3 APC antibody
  11. Mix well and incubate for 30 minutes in the dark in the refrigerator (2-8 °C).
  12. Wash cells twice by adding 2 mL of cold PBS buffer and centrifuge at 400 g for 5 minutes at 4 °C. Aspirate/decant supernatant completely.
  13. Add 300 μL of 1 % formaldehyde in PBS.

Store the stained samples at 2-8 °C until measured in flow cytometer. Analyze the processed samples within 4 hours.

Flow Cytometry:

Analyze the stained samples using flow cytometer equipped with the 488 nm and 633 nm excitation lasers and an appropriate filter set-up. Refer to your cytometer specifications to identify the detectors in which the fluorescence of the stained cells will be collected. Set the voltage on light scatter detectors, forward angle light scatter (FSC) and side (perpendicular) light scatter (SSC) so that the events of interest are on scale. Set the threshold on FSC so that only cells of interest are recorded and most of the debris excluded. Set the voltage on fluorescence detectors so that all measured events are on scale. Acquire at least 20 000 - 30 000 CD4+ lymphocytes per sample. Acquire data using software intended for sample analysis supplied with cytometer.

结果分析

Leukocyte scatter plot:
Visualize measured data as a dot-plot, where forward-scatter (FSC) is on the X-axis and side-scatter (SSC) is on the Y-axis (Figure 1). Set the target cell population (lymphocytes) gate and threshold. Then visualize events from the lymphocyte gate as a dot-plot, where the X-axis represents forward-scatter area (FSC-A) and the Y-axis represents forward-scatter height (FSC-H). Separate the singlets using a diagonal gate.

CD4+ T cells:
Visualize the lymphocyte singlets as a dot-plot, where X-axis represents fluorescence intensity in FITC detector and the Yaxis represents side-scatter (SSC). Carefully set the gate around CD4+ lymphocytes.

CD4+CD25+FOXP3+ Treg cells:
Visualize CD4+ lymphocytes as a dot-plot, where the X-axis represents the FOXP3 signal (fluorescence intensity in APC fluorescence detector) and the Y axis represents CD25 signal (fluorescence intensity in PE fluorescence detector). Place the gate that separates the CD4+CD25+FOXP3+events. Use appropriate controls (APC- and PE-labelled isotype antibodies) to set the discrimination lines correctly.

限制 仅限研究用
储存条件 4 °C