Identify Th1 Cells human IL-2
producing cells
PerCP/Cy5.5 Anti-human IL-2 PE Anti-human CD3
PMA + ionomycin-stimulated (6 hours) human peripheral blood lymphocytes intracellular stained with MQ1-17H12 PerCP/Cy5.5 and CD3 (UCHT1) PE
Anti-human IL-17 Alexa Fuor® 647/CD3 FITC/CD4 PE cocktail
CD3 FITC
PMA/ionomycin-stimulated (6hr) human peripheral blood lymphocytes stained with Human Th17 flow(TM) (one-step) kit. Dot plot analysis is derived from gated in CD3+ cell population.
SSC
Forward Light Detector Cell Surface Area
FSC
前向角散射光（FSC, Forward Scatter） 细胞相对大小及其表面积 侧向角散射光（SSC, Side Scatter） 细胞粒度及细胞内相对复杂性
外周全血细胞散射光双参数点图 （红细胞溶解后）
研究对象为生物颗粒，如各种细胞、微生物及人 工合成微球等 研究的微粒特性包括多种物理及生物学特征，并 加以定量


光学系统
散射光信号
Right Angle Light Detector Cellensor
Incident Light Source
Treg细胞鉴定
Treg
T regulatory (Treg) cells are a subset of T lymphocytes which is usually characterized by CD4+/CD25+/FOXP3+. These naturally occurring Treg cells originate in the thymus, and comprise 2-10% of peripheral CD4+ T cells.
Identify Th17 Cells mouse IL-17 producing cells
Alexa Fluor® 647 Anti-mouse IL-17A, Clone TC11-18H10.1
PMA+Ionomycin-stimulated mouse thymoma cell line EL-4 intracellular stained with TC1118H10.1 Alexa Fluor® 647
Th17细胞鉴定
Th17 cells are a subset of CD4+ T cells defined by their ability to produce the cytokines IL-17, IL-21 and IL-22.
Identify Th17Cells human IL-17 producing cells
T Cell Receptors
Categories - Cells
B Cells (Breg, etc) Dendritic Cells Granulocytes (Basophils, Eosinophils, Neutrophils) Monocytes/Macrophages NK Cells Stem Cells T Cells (TCRs, Th, Treg, etc)


B: Treg flow kit
1.anti-FOXP3 antibody 2.Isotype control for FOXP3 3.FOXP3 Fix/Perm puffer 4.FOXP3 Perm buffer 5.anti-CD4/CD25 antibody Cocktail
Th1/Th2鉴定
Identify Th1 Cells human IFN-γ producing cells
Alexa Fluor® 700 Anti-human IFN-γ PE Anti-human CD3
PMA+ionomycin-stimulated (5 hours) human PBMCs surface stained with CD3 PE and intracellular stained with B27 Alexa Fluor® 700
Forward Scatter
Side Scatter
数据分析
表面标记
胞内标记
流式荧光选择
常用的标记单克隆抗体的荧光素


488nm激光激发： FITC（异硫氰酸荧光素）： 荧光强度可受pH的影响，当pH降低 时其荧光强度也随之减弱 PE（藻红蛋白） PerCP（多甲藻叶绿素蛋白）：光量子产量并不太高，最好应用 在检测表达较高的抗原上 PerCP-Cy5.5（叶绿素蛋白偶联物） PE-Cy7（藻红蛋白偶联物） PE-Cy5（藻红蛋白偶联物，又称Cy-Chrome） PE-Texas Red（藻红蛋白-德州红偶联物，又称ECD） 633nm激光激发： APC（别藻青蛋白） APC-Cy7（别藻青蛋白偶联物）
白细胞表面常用的标记
流式应用(部分 )
Categories - Biological Areas
Adhesion Molecules CDs and Related Molecules Costimulatory Molecules Cytokine & Chemokine and their Receptors Innate Immunity MHC Antigens Nuclear Transcription Factors
Identify Th2 Cells mouse IL-10 producing cells
PE Anti-mouse IL-10 PE/Cy5 Anti-mouse CD4
PMA/Ionomycin-stimulated BALB/c mouse T cells were surface stained with CD4 PE/Cy5 and then intracellular stained with JES5-16E3 PE
Identify Th1 and Th2Cells Panels
Human Th1-Th2 Cytokine Flow Panel, 25 tests
Mouse Th1-Th2 Cytokine Flow Panel, 25 tests
The Th1-Th2 cytokine flow panel is composed of pre-titration tested fluorochromeconjugated anti-cytokine mAbs (FITC anti-IL-2, FITC anti-IFN-γ, PE anti-IL-4, and PE-anti-IL-10) and fixation and permeabilization buffers. This panel of products can be used for evaluation of Th1/Th2 cell populations based on intracellular cytokine staining and flow cytometric analysis.
Identify T Reg Cells human FoxP3-CD4-CD25_3
Alexa Fluor® 488 anti-human FOXP3/CD25 PE/CD4 PerCP antibody cocktail (FOXP3 ,Clone 206D)
Human peripheral blood mononuclear cells stained with One Step Staining Human Treg FlowTM Kit (FOXP3 Alexa Flour® 488/CD25 PE/CD4 PerCP )
Fluorochrome and Instrument

荧光选择方案
Epitope Density Low Medium High
Fluorochrome PE, APC Tandem Dyes FITC, Pacific Blue ™ , Alexa Fluor® 700
Immunity, Volume 28, Issue 5, Pages 639-650 by Tom Tedder’s group in Duke “A Regulatory B Cell Subset with a Unique CD1dhiCD5+ Phenotype Controls T Cell-Dependent Inflammatory Responses”
流式细胞术及其应用
主要内容
流式原理 流式应用


Treg cells Th17 cells Th1/Th2 Breg cells Flowcytomix Tetramer





流式细胞术的基本概念

流式细胞术（Flow Cytometry, 简称FCM）是一 种可以快速、准确、客观，并且同时检测单个微 粒（通常是细胞）的多项特性，并加以定量的技 术，同时可以对特定群体加以分选
Breg鉴定
Identify Breg (B10)Cells
Adopted from The Journal of Immunology, 2006, 176: 705-710.