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. |
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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 |
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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。