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流式辅助试剂、工具
流式细胞技术分析的过程中，有很多因素会影响实验结果：样本的处理、荧光补偿的调节、Fc受体的阻断、活细胞和死细胞的鉴别等。
eBioscience公司提供流式染色缓冲液、细胞固定液、细胞破膜剂、细胞刺激剂、细胞阻断剂、FcR抗体、红细胞裂解液、细胞活性染料、细胞计数微球、荧光补偿微球等。为您的流式实验提供更稳定的结果。
流式Buffer
Intracellular Staining Buffer Selection | ||
Antigen | Foxp3 Staining Buffer Set | Fixation & Permeabilization |
HUMAN |
|
|
IFNy | * | YES |
IL-1a | nt | YES |
IL-1b | nt | YES |
IL-2 | nt | YES |
IL-4 | * | YES |
IL-5 | nt | YES |
IL-6 | nt | YES |
IL-9 | nt | YES |
IL-10 | * | YES |
IL-12 | nt | YES |
IL-13 | nt | YES |
IL-17A | YES | YES |
IL-17F | nt | YES |
IL-21 | * | YES |
IL-22 | * | YES |
Granzyme B | nt | YES |
Perforin | nt | YES |
TNFalpha | YES | YES |
Eomes | YES | NO |
Foxp3 | YES | NO |
Gata-3 | YES | NO |
RORy(t) | YES | NO |
T-Bet | YES | NO |
|
|
|
MOUSE |
|
|
IFNy | YES | YES |
IL-1b | nt | YES |
IL-2 | YES | YES |
IL-4 | * | YES |
IL-6 | nt | YES |
IL-10 | * | YES |
IL-17A | YES | YES |
IL-17F | YES | YES |
IL-21 | NO | YES |
IL-22 | * | YES |
Granzyme B | YES | YES |
Perforin | YES | YES |
TNFalpha | YES | YES |
Eomes | YES | NO |
Foxp3 | YES | NO |
Gata-3 | YES | NO |
Nanog | YES | NO |
Oct 3/4 | YES | NO |
RORy(t) | YES | NO |
T-Bet | YES | NO |
nt = Not tested YES = Staining compatible with this buffer system NO = Staining not compatible with this buffer system *Staining is possible with the Foxp3 Staining Buffer Set, although in some cases a reduction in brightness has been observed. No change in the percentage of positive cells has been observed. | ||
| ||
磷酸蛋白特异性流式抗体Buffer选择指南
Target | Clone | IC fix/perm | Foxp3 | IC fix/methanol |
pSTAT1 (Y701) | KIKSI0803 | - | - | +++ |
pSTAT4 (Y693) | 4LURPIE | - | - | +++ |
pSTAT5 (Y694) | SRBCZX | - | - | +++ |
pSTAT6 (Y641) | CHI2S4N | - | - | +++ |
pSrc (Y418) | SC1T2M3 | ++ | ++ | +++ |
pZAP-70/SYK Y319/Y352 | n3kobu5 | ++ | ++ | +++ |
pSYK (Y348) | moch1ct | ++ | ++ | +++ |
pBTK/ITK (Y551/Y511) | M4G3LN | ++ | ++ | +++ |
pERK1/2 (T202/Y204) | MILAN8R | - | - | +++ |
pNFkB p65 (S529) | B33B4WP | +++ | - | +++ |
pS6 (Y235/Y236) | cupk43k | +++ | ++ | +++ |
pH2AX (S139) | CR55T33 | - | +++ | +++ |
细胞刺激/阻断剂
细胞刺激：未活化的细胞所分泌的细胞因子很少，用流式细胞术很难检测出来，因此在检测前需要刺激淋巴细胞使其活化。eBioscience提供多种细胞刺激物：ConA、LPS、PMA、PHA-L
细胞因子转运抑制剂： 细胞在活化后，分泌的细胞因子会转运到细胞外。流式检测只能对细胞表面或细胞内部的抗原进行检测，因此必须阻止细胞因子分泌到细胞外，使其聚集在细胞内。常用的阻断剂有BFA和Monensin。
Mouse Cytokines: Intracellular Staining Quick Guide | ||||||
Mouse Cytokine | Cell Source | Activation | Incubation Time | Restimulation | Intracellular Block | Antibody |
GM-CSF | mouse spleen | ConA (3ug/mL) (2d)/IL-2 (20ng/mL)+IL-4 (20ng/mL) (3d) | 2d/3d | anti-CD3 (10ug/mL immobilized) + anti-CD28 (2ug/mL soluble) 5hr | Brefeldin A | MP1-22E9 |
IFN-gamma | mouse spleen | ConA (3ug/mL) (2d)/IL-2 (20ng/mL)+IL-4 (20ng/mL) (3d) | 2d/3d | anti-CD3 (10ug/mL immobilized) + anti-CD28 (2ug/mL soluble) 5hr | Brefeldin A | XMG1.2 |
IL-1 alpha | mouse PEC | mIFNγ (100ng/mL) (2hr)/LPS (100ng/mL)(22hr) | 2hr/22hr | - | Brefeldin A | ALF-161 |
IL-1 beta | mouse PEC | LPS (100 ng/mL) (22hr) | 22hr | - | Monensin | NJTEN3 |
IL-2 | mouse spleen | ConA (3ug/mL) (2d)/IL-2 (20ng/mL)+IL-4 (20ng/mL) (3d) | 2d/3d | anti-CD3 (10ug/mL immobilized) + anti-CD28 (2ug/mL soluble) 5hr | Brefeldin A | JES6-5H4 |
IL-4 | mouse spleen | Th2 polarized | 6d | PMA (50 ng/mL) + Ionomycin (1 ug/mL) 5hr | Brefeldin A | BVD6-24G2, |
IL-5 | mouse splenic CD4 | ConA (3ug/mL) (2d)/IL-2 (20ng/mL)+IL-4 (20ng/mL) (3d) | 2d/3d | immobilized) + anti-CD28 (2ug/mL soluble) 5hr | Brefeldin A | TRFK5 |
IL-6 | mouse PEC | LPS (100 ng/mL) (22hr) | 22hr | - | Monensin | MP5-20F3 |
IL-10 | mouse spleen | ConA (3ug/mL) (2d)/IL-2 (20ng/mL)+IL-4 (20ng/mL) (3d) | 2d/3d | anti-CD3 (10ug/mL immobilized) + anti-CD28 (2ug/mL soluble) 5hr | Brefeldin A | JES5-16E3, |
IL-12/IL-23 (p40) | mouse PEC | LPS (100 ng/mL) (22hr) | 22hr | - | Brefeldin A | C17.8 |
IL-13 | mouse spleen | Th2 polarized | 6d | PMA (50 ng/mL) + Ionomycin (1 ug/mL) 5hr | Brefeldin A | eBio13A |
IL-17A | mouse spleen | Th17 polarized | 6d | PMA (50 ng/mL) + Ionomycin (1 ug/mL) 5hr | Monensin | eBio17B7 |
IL-17F | mouse spleen | Th17 polarized | 6d | PMA (50 ng/mL) + Ionomycin (1 ug/mL) 5hr | Monensin | eBio18F10 |
IL-21 | mouse spleen | Th17 polarized | 9d | PMA (50 ng/mL) + Ionomycin (1 ug/mL) 5hr | Monensin | FFA21 |
IL-22 | mouse spleen | Th17 polarized | 12d | PMA (50 ng/mL) + Ionomycin (1 ug/mL) 5hr | Brefeldin A | IL22JOP |
IL-23 p19 | mouse bone marrow | mGM-CSF (40 ng/mL) | 8d | LPS (1 ug/mL) (24 hr) | Monensin | fc23cpg |
MCP-1/ CCL2 | mouse PEC | LPS (100 ng/mL) (24hr) | 24hr | - | Brefeldin A | 2H5 |
TNF alpha | mouse spleen | ConA (3ug/mL) (2d)/IL-2 (20ng/mL)+IL-4 (20ng/mL) (3d) | 2d/3d | anti-CD3 (10ug/mL immobilized) + anti-CD28 (2ug/mL soluble) 5hr | Brefeldin A | MP6-XT22, |
Annotations: mouse PEC=mouse thioglycolate-elicited peritoneal macrophages; ConA=Concanavalin A; Iono=Ionomycin; LPS=Lipopolysaccharide; PMA=Phorbol Myristate Acetate; 2d=2 day culture; 5hr=5 hour culture | ||||||
Human Cytokines: Intracellular Staining Quick Guide | ||||||
Human Cytokine | Cell Source | Activation | Incubation Time | Restimulation | Intracellular Block | Antibody |
G-CSF | PBMC | LPS (1 ug/mL) | 24hr | - | Monensin | 8F5CSF |
GM-CSF | PBMC | PMA (30-50ng/mL)/Iono (1ug/mL) | 5hr | - | Monensin | BVD2-21C11 |
IFN gamma | PBMC | PMA (30-50ng/mL)/Iono (1ug/mL) | 5hr | - | Brefeldin A | 4S.B3 |
IL-1 alpha | PBMC | LPS (1 ug/mL) | 24hr | - | Monensin | 364/3B3-14, CRM8 |
IL-1 beta | PBMC | LPS (100ng/mL) | 4hr | - | Brefeldin A | CRM56 |
IL-1RA | PBMC | LPS (100ng/mL) | 24hr | - | Brefeldin A | CRM17 |
IL-2 | PBMC | PMA (30-50ng/mL)/Iono (1ug/mL) | 4-6hr | - | Brefeldin A | MQ1-17H12 |
IL-4 | PBMC | PMA (30-50ng/mL)/Iono (1ug/mL) | 4-6hr | - | Brefeldin A | 8D4-8 |
IL-5 | CD4 | Th2 polarizing cultures | 6d | PMA (50ng/mL) + Ionomycin (1ug/mL) (5hr) | Brefeldin A | TRFK5, JES1-5A10 |
IL-6 | PBMC | LPS (100ng/mL) | 24hr | - | Brefeldin A | MQ2-13A5 |
IL-9 | CD4 | Th2 polarizing cultures | 6d | PMA (50ng/mL) + Ionomycin (1ug/mL) (5hr) | Monensin | MH9A4 |
IL-10 | CD4 | Th2 polarizing cultures | 6d | PMA (50ng/ml) + Ionomycin (1ug/ml) (5hr) | Monensin | JES3-9D7 |
IL-12/ IL-23 (p40) | PBMC | hIFN gamma (100ng/mL) (2hr)/LPS (100ng/mL) (22hr) | 2hr/22hr | - | Brefeldin A | C8.6 |
IL-13 | CD4 | anti-CD3 (10ug/mL, immobilized) + anti-CD28 (2ug/mL, soluble) + IL-2 (10ng/mL) + IL-4 (20ng/mL) (2d); IL-2 (10ng/mL) + IL-4 (20ng/mL) (3d) | 2d/3d | PMA (5ng/ml) + Ionomycin (500ng/ml) (4hr) | Brefeldin A | PVM13-1 |
IL-17A | PBMC | Th17 polarizing cultures | 6d | PMA (50ng/ml) + Ionomycin (1ug/ml) (5hr) | Brefeldin A | eBio64CAP17, eBio64DEC17 |
IL-21 | PBMC | PMA (30-50ng/mL)/Iono (1ug/mL) | 4-7hr or 12-18hr | - | Brefeldin A | eBio3A3-N2 |
IL-22 | CD4 | Th17 polarizing cultures | 6d | PMA (50ng/ml) + Ionomycin (1ug/ml) (5hr) | Brefeldin A | IL22JOP |
IL-23 p19 | PBMC | hGM-CSF (40ng/mL) +hIL-4 (40ng/mL) | 6d | LPS (1ug/mL) (24hr) | Monensin | 23dcdp |
MCP-1/ CCL2 | PBMC | LPS (1ug/mL) | 24hr | - | Monensin | 2H5, 5D3-F7 |
RANTES/ CCL5 | PBMC | LPS (1ug/mL) | 24hr | - | Monensin | VL1 |
TNF alpha | PBMC | PMA (30-50ng/mL)/Iono (1ug/mL) | 5hr | - | Brefeldin A | MAb11 |
TNF beta | PBMC | Th1 polarizing cultures | 6d | PMA (50ng/mL) + Ionomycin (1ug/mL) (5hr) | Monensin | 359-81-11 |
Annotations: Iono=Ionomycin; PMA=Phorbol Myristate Acetate; LPS=Lipopolysaccharide; 2d=2 day culture; 5hr=5 hour culture; LPS for activation of human PBMC obtained from Sigma (#L-8274) | ||||||
Non-Cytokine Proteins: Intracellular Staining Quick Guide | |
Antigen | Antibody |
Mouse/Rat Bcl-2 | 10C4 |
Mouse CTLA-4 (CD152) | UC10-4B9 |
Human CTLA-4 (CD152) | 14D3 |
Human/Mouse/Rat Cytochrome C | 6H2 |
Human Foxp3 | PCH101 |
Mouse Foxp3 | FJK-16s |
GATA3 |
|
Mouse/Human Granzyme B | eBioGrB |
House Langerin (CD207) | eBioRMUL.2 |
Human Nanog | hNanog.1 |
Human PCNA (Proliferatin Cell Nuclear Antigen) | PC10 |
Mouse Perforin | eBioOMAK-D |
Human Perforin | dG9 |
ROR gamma |
|
Mouse SLP-76 | MS76 |
Human SLP-76 | HS76 |
Human/Mouse T-bet | eBio4B10 (4B10, 4-B10) |
Human TLR3 | TLR3.7 |
Mouse TLR9 | M9.D6 |
Human TLR9 | eB72-1665 |
Mouse/Human ZAP-70 | 1E7.2 |
Fc受体阻断剂
Fc受体是指细胞膜表面能与免疫球蛋白（IgG、IgA、IgM、IgE和IgD）Fc片段结合的受体。目前已鉴定明确属于CD抗原的Fc受体有FcγR（CD64、CD32、CD16）、FcαR、FcεR。FcR能够与抗体的Fc段结合，在检测时产生假阳性。 使用FcR阻断剂，可以消除假阳性，降低检测背景，获得更清晰的结果。其广泛存在于NK细胞、肥大细胞、巨噬细胞、中性粒细胞的表面。
用Mouse IgG2a Isotype Control FITC (cat.11-4724) (左图) or Mouse IgG2bIsotype Control FITC (cat. 11-4732) (右图)染U937细胞，没有用Human Fc gamma R-Binding Inhibitor预处理的是紫色底纹，用Human Fcgamma R-Binding Inhibitor预处理的是蓝线，红线是U937细胞自发荧光。
细胞计数：123count eBeads ™
² 在流式平台上计数细胞
² 利用抗体标记细胞并利用123count eBeads计算该细胞群数量
² 可同时并用FVD细胞存活染料标记活细胞计数
应用领域
² 全血细胞计数
² 流式细胞仪计数功能验证
² 精子浓度计数
² 可并用细胞存活染料7-AAD，PI或FVD计数细胞存活率
² 使用磁珠细胞分选试剂盒后计算细胞分选产率与纯度
123counteBeads ™使用方式
• 加入123count eBeads TM
• 选定待计数的细胞
• 公式计算细胞浓度
² 加入 * 已知浓度的123count eBeads 进未知浓度的细胞样本
² 流式细胞仪同时读取细胞与123count eBeads
² 公式计算细胞浓度如下
1. 当细胞与123count eBeads 体积比为1:1混合时
实际细胞数=流式细胞仪显示细胞数量÷流式细胞仪显示eBead数量× eBead 标示浓度
2. 如混合体积比不是1:1时
实际细胞数=（流式细胞仪显示细胞数量 X eBead体积）÷（流式细胞仪显示eBead数量 X 细胞体积）X eBead 标示浓度
*每管123count eBeads TM 标示有该管内eBead的浓度
全血CD4实际细胞计数示范
= (流式细胞仪显示细胞数量/流式细胞仪显示eBead数量) X eBead标示浓度
= (541个细胞/1009 eBeads) X 998 eBeads/ul
= 535个细胞/ul
调节荧光补偿的利器——荧光补偿微球
eBioscience抗体捕获微珠是球状的，可以和荧光抗体结合，作为流式实验的补偿对照。
抗体捕获微珠分为阳性和阴性，阳性微珠能捕获所有的小鼠、大鼠或仓鼠抗体，阴性微珠不会和抗体反应。当荧光共轭抗体加到微珠上，就可以区分出阴阳性微珠，阳性微珠可以作为多色流式实验的单色补偿对照。
优点：
• 通常荧光都比样本表达要强
• CV值小，几乎没有背景荧光
• 和你实际染色的荧光抗体结合
• 不需要使用珍贵的样本
• 使用简单
OneCompeBeads和小鼠、大鼠、仓鼠的免疫球蛋白轻链反应，对488nm蓝色，532nm绿色，561nm黄绿色和633 - 640 nm红色激光所激发的荧光染料的光谱进行补偿。可兼容eFluor® 450，但是对405 nm紫色激发的光谱不能很好的补偿。
UltraComp eBeads和小鼠、大鼠、仓鼠的免疫球蛋白轻链反应，能对355nm紫外，405nm紫色，488nm蓝色，532nm绿色，561nm黄绿色和633 - 640 nm红色激光所激发的荧光染料的光谱进行补偿。
将流式常用的13种不同的PE（见左图），eFluor450（见右图）共轭单克隆抗体和UltraComp eBeads混匀，Beads捕获0.25 ug抗体后上流式细胞仪分析，每个直方图代表一种染色抗体（图右边是克隆和同型）。
实验所需材料：
12x75 mm 圆底试管
直标抗体
流式染色缓冲液 (eBioscience Cat. No.00-4222)
实验操作流程：
准备单色补偿对照
1、 每种荧光染料标记一个管。
2、 微珠通过反复颠倒或涡旋混匀，混匀时间不低于10min。
3、 每个管里加一滴UltraComp eBeads。
4、 每个管里加少量共轭抗体。
5、 用移液枪吸打或涡旋短暂混匀。
6、 2-8℃避光孵育15-30min。
7、 每管加2mL流式染色缓冲液后400-600 ×g离心3-5min。
8、 弃去上清液，加0.2-0.4mL流式染色缓冲液。
9、 分析前用移液枪抽打或涡旋短暂混匀。
