流式輔助試劑、工具
流式細(xì)胞技術(shù)分析的過(guò)程中，有很多因素會(huì)影響實(shí)驗(yàn)結(jié)果：樣本的處理、熒光補(bǔ)償?shù)恼{(diào)節(jié)、Fc受體的阻斷、活細(xì)胞和死細(xì)胞的鑒別等。
eBioscience公司提供流式染色緩沖液、細(xì)胞固定液、細(xì)胞破膜劑、細(xì)胞刺激劑、細(xì)胞阻斷劑、FcR抗體、紅細(xì)胞裂解液、細(xì)胞活性染料、細(xì)胞計(jì)數(shù)微球、熒光補(bǔ)償微球等。為您的流式實(shí)驗(yàn)提供更穩(wěn)定的結(jié)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. | ||
| ||
磷酸蛋白特異性流式抗體Buffer選擇指南
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 | - | +++ | +++ |
細(xì)胞刺激/阻斷劑
細(xì)胞刺激：未活化的細(xì)胞所分泌的細(xì)胞因子很少，用流式細(xì)胞術(shù)很難檢測(cè)出來(lái)，因此在檢測(cè)前需要刺激淋巴細(xì)胞使其活化。eBioscience提供多種細(xì)胞刺激物：ConA、LPS、PMA、PHA-L
細(xì)胞因子轉(zhuǎn)運(yùn)抑制劑： 細(xì)胞在活化后，分泌的細(xì)胞因子會(huì)轉(zhuǎn)運(yùn)到細(xì)胞外。流式檢測(cè)只能對(duì)細(xì)胞表面或細(xì)胞內(nèi)部的抗原進(jìn)行檢測(cè)，因此必須阻止細(xì)胞因子分泌到細(xì)胞外，使其聚集在細(xì)胞內(nèi)。常用的阻斷劑有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受體阻斷劑
Fc受體是指細(xì)胞膜表面能與免疫球蛋白（IgG、IgA、IgM、IgE和IgD）Fc片段結(jié)合的受體。目前已鑒定明確屬于CD抗原的Fc受體有FcγR（CD64、CD32、CD16）、FcαR、FcεR。FcR能夠與抗體的Fc段結(jié)合，在檢測(cè)時(shí)產(chǎn)生假陽(yáng)性。 使用FcR阻斷劑，可以消除假陽(yáng)性，降低檢測(cè)背景，獲得更清晰的結(jié)果。其廣泛存在于NK細(xì)胞、肥大細(xì)胞、巨噬細(xì)胞、中性粒細(xì)胞的表面。
用Mouse IgG2a Isotype Control FITC (cat.11-4724) (左圖) or Mouse IgG2bIsotype Control FITC (cat. 11-4732) (右圖)染U937細(xì)胞，沒(méi)有用Human Fc gamma R-Binding Inhibitor預(yù)處理的是紫色底紋，用Human Fcgamma R-Binding Inhibitor預(yù)處理的是藍(lán)線，紅線是U937細(xì)胞自發(fā)熒光。
細(xì)胞計(jì)數(shù)：123count eBeads ?
2 在流式平臺(tái)上計(jì)數(shù)細(xì)胞
2 利用抗體標(biāo)記細(xì)胞并利用123count eBeads計(jì)算該細(xì)胞群數(shù)量
2 可同時(shí)并用FVD細(xì)胞存活染料標(biāo)記活細(xì)胞計(jì)數(shù)
應(yīng)用領(lǐng)域
2 全血細(xì)胞計(jì)數(shù)
2 流式細(xì)胞儀計(jì)數(shù)功能驗(yàn)證
2 精子濃度計(jì)數(shù)
2 可并用細(xì)胞存活染料7-AAD，PI或FVD計(jì)數(shù)細(xì)胞存活率
2 使用磁珠細(xì)胞分選試劑盒后計(jì)算細(xì)胞分選產(chǎn)率與純度
123counteBeads ?使用方式
? 加入123count eBeads TM
? 選定待計(jì)數(shù)的細(xì)胞
? 公式計(jì)算細(xì)胞濃度
2 加入 * 已知濃度的123count eBeads 進(jìn)未知濃度的細(xì)胞樣本
2 流式細(xì)胞儀同時(shí)讀取細(xì)胞與123count eBeads
2 公式計(jì)算細(xì)胞濃度如下
1. 當(dāng)細(xì)胞與123count eBeads 體積比為1:1混合時(shí)
實(shí)際細(xì)胞數(shù)=流式細(xì)胞儀顯示細(xì)胞數(shù)量÷流式細(xì)胞儀顯示eBead數(shù)量× eBead 標(biāo)示濃度
2. 如混合體積比不是1:1時(shí)
實(shí)際細(xì)胞數(shù)=（流式細(xì)胞儀顯示細(xì)胞數(shù)量 X eBead體積）÷（流式細(xì)胞儀顯示eBead數(shù)量 X 細(xì)胞體積）X eBead 標(biāo)示濃度
*每管123count eBeads TM 標(biāo)示有該管內(nèi)eBead的濃度
全血CD4實(shí)際細(xì)胞計(jì)數(shù)示范
= (流式細(xì)胞儀顯示細(xì)胞數(shù)量/流式細(xì)胞儀顯示eBead數(shù)量) X eBead標(biāo)示濃度
= (541個(gè)細(xì)胞/1009 eBeads) X 998 eBeads/ul
= 535個(gè)細(xì)胞/ul
調(diào)節(jié)熒光補(bǔ)償?shù)睦鳌獰晒庋a(bǔ)償微球
eBioscience抗體捕獲微珠是球狀的，可以和熒光抗體結(jié)合，作為流式實(shí)驗(yàn)的補(bǔ)償對(duì)照。
抗體捕獲微珠分為陽(yáng)性和陰性，陽(yáng)性微珠能捕獲所有的小鼠、大鼠或倉(cāng)鼠抗體，陰性微珠不會(huì)和抗體反應(yīng)。當(dāng)熒光共軛抗體加到微珠上，就可以區(qū)分出陰陽(yáng)性微珠，陽(yáng)性微珠可以作為多色流式實(shí)驗(yàn)的單色補(bǔ)償對(duì)照。
優(yōu)點(diǎn)：
? 通常熒光都比樣本表達(dá)要強(qiáng)
? CV值小，幾乎沒(méi)有背景熒光
? 和你實(shí)際染色的熒光抗體結(jié)合
? 不需要使用珍貴的樣本
? 使用簡(jiǎn)單
OneCompeBeads和小鼠、大鼠、倉(cāng)鼠的免疫球蛋白輕鏈反應(yīng)，對(duì)488nm藍(lán)色，532nm綠色，561nm黃綠色和633 - 640 nm紅色激光所激發(fā)的熒光染料的光譜進(jìn)行補(bǔ)償?？杉嫒輊Fluor? 450，但是對(duì)405 nm紫色激發(fā)的光譜不能很好的補(bǔ)償。
UltraComp eBeads和小鼠、大鼠、倉(cāng)鼠的免疫球蛋白輕鏈反應(yīng)，能對(duì)355nm紫外，405nm紫色，488nm藍(lán)色，532nm綠色，561nm黃綠色和633 - 640 nm紅色激光所激發(fā)的熒光染料的光譜進(jìn)行補(bǔ)償。
將流式常用的13種不同的PE（見(jiàn)左圖），eFluor450（見(jiàn)右圖）共軛單克隆抗體和UltraComp eBeads混勻，Beads捕獲0.25 ug抗體后上流式細(xì)胞儀分析，每個(gè)直方圖代表一種染色抗體（圖右邊是克隆和同型）。
實(shí)驗(yàn)所需材料：
12x75 mm 圓底試管
直標(biāo)抗體
流式染色緩沖液 (eBioscience Cat. No.00-4222)
實(shí)驗(yàn)操作流程：
準(zhǔn)備單色補(bǔ)償對(duì)照
1、 每種熒光染料標(biāo)記一個(gè)管。
2、 微珠通過(guò)反復(fù)顛倒或渦旋混勻，混勻時(shí)間不低于10min。
3、 每個(gè)管里加一滴UltraComp eBeads。
4、 每個(gè)管里加少量共軛抗體。
5、 用移液槍吸打或渦旋短暫混勻。
6、 2-8℃避光孵育15-30min。
7、 每管加2mL流式染色緩沖液后400-600 ×g離心3-5min。
8、 棄去上清液，加0.2-0.4mL流式染色緩沖液。
9、 分析前用移液槍抽打或渦旋短暫混勻。
