磁珠分选原理和应用课件-在线预览

Miltenyi

Biotec

stands

for

MACS

Technology.MACS

stands

for

Magnetic

Cell

Sorting磁珠分选原理和应用There

are

a

lot

of

different

separation

techniques

apart

from

Magnetic

Cell

Sorting.Some

of

themare

also

used

to

pre-enrich

cells

befor

performing

a

megnetic

cell

sorting.内容MACS技术原理MACS技术设备及分选策略几种特殊的MACS分选分选和分析DC细胞分选各种T细胞分选和分析细胞因子分泌细胞分选造血干细胞分选人类肿瘤细胞分选和分析凋亡和死亡细胞分选转染细胞分子生物学和蛋白生化的分选M

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organisms

such

as

man

bears

many

different

cell

types.

Here,

different

blood

cell

types

are

shown,

all

develpingfroma

central

stemcell.

Foralot

ofapplications

it

is

necessary

to

obtain

one

single

cell

type.MACS技术原理MACS(Magnetic

Activated

Cell

Sorting)：=免疫学+细胞生物学+磁力学基于抗体对抗原的特异性识别磁性微珠直接或者间接偶联在抗体上，从而与细胞相连在高强度、梯度磁场中达到细胞磁性分离的目的M

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1One

cell

type

in

amixture

is

labeled

with

aspecific

monoclonal

antibody

coupled

to

a

verysmall

magnetic

bead.

The

cells

are

passed

over

a

column

which

is

placedin

the

magnetic

field

of

a

MACS

separator.

The

magnetically

labeled

cells

are

retained

in

the

column,

the

unlabeld

cells

run

through.

Then,

the

column

is

removedfrom

the

magnet

and

the

labeled

cells

are

eluted

as

the

positive

fraction.

We

call

this

a

positive

selection.MACS®磁性分选MACS微珠进行磁性标记未标记的细胞先行流出洗脱阳性分选的细胞M

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1There

are

a

lot

of

different

separation

techniques

apart

from

Magnetic

Cell

Sorting.Some

of

themare

also

used

to

pre-enrich

cells

befor

performing

a

megnetic

cell

sorting.分选前标本制备：过滤MACS预选滤器：30

mM

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7

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are

a

lot

of

different

separation

techniques

apart

from

Magnetic

Cell

Sorting.Some

of

themare

also

used

to

pre-enrich

cells

befor

performing

a

megnetic

cell

sorting.MACS技术设备与试剂»MACS微珠»MACS分选柱»MACS分选器»详细分选方案*M

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see

the

MicroBeads

you

have

to

make

a

cross-section

through

the

cell

and

performa

transmission

electron

micrograph.

The

MicroBeads

are

sittingon

thesurface

(arrows).

You

can

also

see

the

douple

membrane

of

the

cell.M

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3MACS技术分选的CD8+T细胞微珠MACS微珠直标微珠多选微珠间标微珠MACS技术设备与试剂MACS分选柱：》填充有不同规格的铁珠。》铁珠表面有亲水包被，不损伤细胞。》无菌包装。》可用于分选多种细胞及亚细胞物质、细菌、病毒、mRNA和蛋白质。专利产品将磁场扩大1000－10000倍——高纯度，高回收率Reason

for

the

steel

balls:

They

generate

a

high

gradient

magnetic

field

withextremely

strong

magnetic

forces

in

the

„edges“.

That

allows

MiltenyiBiotecto

putextremely

few

material

on

the

cell

surface,

i.e.

the

tiny

MicroBeads.M

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take

home

(or

better

„take

to

the

customer“

messages

which

are

explained

in

the

following

slides.MACS技术主要优势小磁珠:50

nm（快速、不活化细胞、不沉淀）

保留细胞功能（直接进行后续实验；无毒性；氧化铁和多聚糖组成，可生物降解）

与流式细胞仪兼容（不影响细胞光学特性、不影响荧光抗体标记、与流式细胞仪分选联合使用可以缩短流式分选时间）快速、简便、无菌

适用范围广（磁珠种类多、从少量到大量、适用于分选各种细胞、亚细胞物质）M

D

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of

different

MACS

separation

strategies:

no

limit

in

cell

separation!MACS®细胞分选策略•阳性分选去除分选去除分选后再阳性分选多重分选M

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2阳性分选策略MACS®微珠磁性标记未标记细胞先行流出将分选柱移出磁场，洗脱阳性分选细胞MACS细胞分选策略2、去除分选：•去除分选是磁性标记非目的细胞，并将其从细胞混合物中去除，即未磁性标记的细胞为目的细胞。去除分选策略适用范围：》去除不需要的细胞》缺乏针对目的细胞的特异性抗体（如肿瘤细胞）》不需要抗体和目的细胞结合》需进一步通过阳性选择分选细胞亚群M

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other

cells

are

labeled

withantibodies

(could

be

indirect

as

shownhere

or

direct.)Some

customers

prefer

this

method,

althoughit

is

more

difficult

to

obtain

the

cells

you

would

like

to

habe

(you

label

the

cells

which

you

do

not

want)

and

it

is

alsomore

expensive

(more

antibodies,

more

MicroBeads).

In

doubt,

Positive

Selection

should

be

the

method

of

choice.去除分选磁性标记分选未磁性标记细胞M

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21.去除非CD4+T细胞。用生物素化抗体混合物和抗生物素微珠间接磁性标记非CD4+T细胞磁性分选去除标记细胞先行流出为CD4+T细胞2.CD25微珠直接磁性标记CD25+细胞阳性分选靶细胞CD4+CD25+调节性T滞留在分选柱上，离开磁场后洗脱下来去除后再阳性分选CD4+CD25+双阳性细胞To

isolate

a

cell

according

to

two

different

markers:

A

positive

selection

followed

by

a

second

positive

selection多选微珠标记MACS分选磁性颗粒的酶解离终止解离反应M

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1根据第二标志进行磁珠标记MACS分选MACS细胞分选策略4、多选微珠：What

is

possible

for

cells,

is

also

possible

for

molecules.M•ACSM畅ini销MAC机S

s器tar与tin分g

k选it：柱1个分选架s1个MiniMACS分选器s1盒MS分选柱s1支价值7500元试剂（15088元）：可做阳性分选s小量标本s间标去除分选s分子生物学分选Mini&

MidiMACS

starting

kit：1个分选架s1个MiniMACS分选器s

1个MidiMACS分选器s1盒

MS分选柱s

1盒LS/LD分选柱s1支价值7500元试剂（27838元）：可做阳性分选和去除分选MS分选柱：1盒25个，2763元LS分选柱：1盒25个，4356元LD分选柱：1盒25个，5100元uMACS与u或者M分选柱：分子生物学分选Vario

MACS分选仪auto

MACS自动分选仪sCliniMACS分选仪M

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is

possible

for

cells,

is

also

possible

for

molecules.MACS几种畅销磁珠与抗体专利产品：CD133sCD303sCD304•特殊产品：人类DC系列：BDCA-1(CD1c)sBDCA-2(CD303)sBDCA-3(CD141)sBDCA-4(CD304)小鼠DC系列：m

PDCA微珠与抗体干细胞产品：CD133sCD271微珠与抗体细胞因子分泌细胞：CSA分选与分析TH2细胞：

CD294(CRTH)微珠与抗体分选人类肿瘤细胞：上皮肿瘤：CKsHEA非血液肿瘤：CD45去除死亡细胞去除试剂盒分选转染细胞分选HIV病毒sm

RNAscDNAtop

ten：CD133sCD34s人CD4+CD25+s小鼠CD4+CD25+ CD303/CD304s人类NKs小鼠CD11csanti-PE微珠 小鼠干细胞试剂盒（系别阴性＋CD117）sCD4/CD8M

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is

possible

for

cells,

is

also

possible

for

molecules.主要竞争对手分析Dynal：大磁珠主要市场：CD4、CD8等高频细胞，优势：价格劣势：回收率低（不能做低频细胞、弱表达细胞分选）磁珠必需剪切（耗时——细胞状态不佳）大磁珠（活化细胞）我们的优势：纯度高、回收率高、细胞活性好不活化细胞，不改变细胞功能省时、省细胞产品品种最多，有专利产品有手动、自动、临床级产品有强大的技术支持队伍很好的声誉，市场占有率最高M

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is

possible

for

cells,

is

also

possible

for

molecules.主要卖点干细胞产品：人类：CD133、CD34、MSC试剂盒（CD271）小鼠：系别阴性试剂盒、CD117微珠定向分化培养基DC产品：

人类：BDCA-1、BDCA-2/4小鼠：CD11cNK产品：

人类：NK细胞阴选试剂盒、CD3/CD56T细胞产品：

CD4+CD25+调节性T细胞（人和小鼠）CSA系列CD4、CD8微珠及阴选试剂盒B细胞产品：CD19微珠及阴选试剂盒肿瘤细胞分选：癌细胞富集试剂盒、HEA微珠、CD45阴选单核细胞分选：CD14微珠及阴选试剂盒间标微珠死细胞去除试剂盒预选滤器、FCR阻断剂、T细胞活化与扩增试剂盒流式细胞仪抗体（价廉、包装量大、优质）M

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2识别DC和DC亚群NewLineage-

(CD3,

CD14,

CD16,

CD19,

CD20,

CD56)

HLA-DR+PDCCD11c-

CD123+BDCA-2(CD303)+BDCA-4(CD304)+CD123lowCD123-CD11c+

CD123low/-MDCMDC1MDC2BDCA-1(CD1c)+BDCA-3(CD141)+

+DC亚群特异性标志:Blood

DendriticCell

AntigensBDCA-2/4+BDCA-1+BDCA-3+CD11c+

DCCD8+

DCCD4+

DCCD11c+

B220+

Gr-1(Ly-6C/G)+DCCD11c+

B220-

DCConventional

DC(“lymphoid“

DC)(“myeloid“

DC)CD11c

CD8aCD11c

CD4PEFITCAPCCD11cCD11c

B220

Gr-1Plasmacytoid

DC

(PDC)mPDCA-1MACS®技术分选与分析小鼠DC细胞NewCytokine

Secretion

Assay

is

a

completelynew

technology

(gota

different

colour

on

the

slide).

Analysis

and

also

enrichment

of

extremely

rare

antigen-specificTcells,

for

example.MACS®技术分选与分析大鼠DC细胞NewAnti-DC(OX62)微珠用于从大鼠外周血、淋巴和非淋巴组织中分选或者去除OX62+DC。单克隆抗体OX62识别表达于大部分大鼠DC上的整合素αE2

单位（分子量亚150kDa）。两个主要的树突状细胞亚群：CD4/OX41双阳性和CD4/OX41双阴性亚群。该抗原也表达于表皮TCRα/β+或γ/δ+的T细胞亚群和垂体前叶星状滤泡样（S100+）细胞的一个亚群，但在朗格罕氏细胞和巨噬细胞上不表达。M

D

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Secretion

Assay

is

a

completelynew

technology

(gota

different

colour

on

the

slide).

Analysis

and

also

enrichment

of

extremely

rare

antigen-specificTcells,

for

example.MACS®技术分选与分析功能性T细胞M

D

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restimulation

leads

to

cytokine

secretionCatch

reagent

forms

an

artificial

surface

to

catch

the

cytokine

which

is

secreted.

Catch

reagent

is

composed

of

two

antibodies,

one

of

themdirected

against

thecytokine.

Labeling

with

detectionantibody.

Indirect

magnetic

labeling

is

optional

for

enrichment.Without

enrichment

(no

columns):

detect

frequencies

between

0.01-0.1%of

total

cells.Withenrichment:

detect

frequencies

down

to

0.0001

%

(1

cell

in

10e6

cells!!)In

addition,

cells

stay

alive

duringenrichment

and

detection

and

can

be

taken

into

culture

or

used

for

molecular

analysis.MACS®细胞因子分泌细胞分析与分选New抗原特异性再刺激细胞因子分泌阶段捕获试剂标记荧光标记和磁性分选••••Catch

ReagentM

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2Detection

AntibodyAnti-PEMicroBeadscytokinesecretingcellsecretedcytokineAntigen-specific

restimulation

leads

to

cytokine

secretionCatch

reagent

forms

an

artificial

surface

to

catch

the

cytokine

which

is

secreted.

Catch

reagent

is

composed

of

two

antibodies,

one

of

themdirected

against

thecytokine.

Labeling

with

detectionantibody.

Indirect

magnetic

labeling

is

optional

for

enrichment.Without

enrichment

(no

columns):

detect

frequencies

between

0.01-0.1%of

total

cells.Withenrichment:

detect

frequencies

down

to

0.0001

%

(1

cell

in

10e6

cells!!)In

addition,

cells

stay

alive

duringenrichment

and

detection

and

can

be

taken

into

culture

or

used

for

molecular

analysis.MACS®细胞因子分泌细胞分析与分选New分选前M

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2刺激组分选后未刺激组*百分比均为占CD4＋细胞百分比Antigen-specific

restimulation

leads

to

cytokine

secretionCatch

reagent

forms

an

artificial

surface

to

catch

the

cytokine

which

is

secreted.

Catch

reagent

is

composed

of

two

antibodies,

one

of

themdirected

against

thecytokine.

Labeling

with

detectionantibody.

Indirect

magnetic

labeling

is

optional

for

enrichment.Without

enrichment

(no

columns):

detect

frequencies

between

0.01-0.1%of

total

cells.Withenrichment:

detect

frequencies

down

to

0.0001

%

(1

cell

in

10e6

cells!!)In

addition,

cells

stay

alive

duringenrichment

and

detection

and

can

be

taken

into

culture

or

used

for

molecular

analysis.MACS®细胞因子分泌细胞分析New刺激组未刺激组使用PE-CMVpp65/HLA/A2-tetramers和IFN-g

Secretion

Assay-Detection

Kit

(APC)or

IL-2

Secretion

Assay-

Detection

Kit

(APC)检测HLA/A2

CMV+患者PBMC。*百分比均为占CD8＋细胞百分比M

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2人类细胞因子分泌细胞分选与检测试剂盒IFN-g分泌细胞IL-2分泌细胞IL-4分泌细胞IL-10分泌细胞TNF-

分泌细胞MACS®细胞因子分泌细胞分析与分选New小鼠细胞因子分泌细胞分选与检测试剂盒•••••IFN-g分泌细胞IL-2分泌细胞IL-4分泌细胞IL-5分泌细胞IL-10分泌细胞What

is

possible

for

cells,

is

also

possible

for

molecules.人类干细胞分选微珠与试剂盒CD34微珠及多选微珠：用于从骨髓、外周血、脐带血中分选造血干细胞、内皮前体细胞CD133微珠：用于从骨髓、外周血、脐带血中分选造血干细胞、内皮前体细胞、多能成体干细胞（MAPC）

CD117微珠：用于从骨髓、外周血、脐带血中分选造血干细胞、骨髓间充质细胞CD45/GlyA去除分选试剂盒：用于从骨髓中阴选分选多能成体干细胞（MAPC）CD105微珠：用于从骨髓中分选间充质细胞

抗成纤维细胞微珠：用于从骨髓中分选间充质细胞多能成体干细胞（MAPC）MSC分选工具（CD271）new：用于从骨髓中分选间充质细胞M

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2NBTstains

for

alkaline

phosphataseIntroduction:In

bone

marrow

(BM)

stemand

progenitor

cells

withhaematopoietic

differentiationpotential

exist

in

close

proximity

to

the

heterogeneous

group

of

stemcells

withnonhaematopoietic

(NH)

differentiationpotential.

Marrow

stromal

cells

(MSCs)

with

the

ability

to

strongly

adhere

to

plastic

comprise

a

heterogeneous

population

ofNH

stemcells

with

broad

plasticity.

Many

attempts

have

been

made

to

find

the

most

efficient

marker

for

the

isolation

of

these

MSCs,

such

as

Stro-1,

CD117,CD105,

CD271,

and

anti-fibroblast

antigen.

CD271+

cells

were

reported

to

showa

one

to

three

log

greater

expansion

in

number

of

fibroblastic

cells

and

agreatercapacity

to

give

rise

to

adipocyte

colonies

and

induce

osteoblastic

differentiation,

therefore

CD271

has

become

one

of

the

most

promising

and

distinct

marker

forMSC

isolation

(Abstract

Jones

04/Quirici02).MSC

Research

Tool

Box

-

CD271(LNGFR)new成分:分选:CD271

(LNGFR)-APCAnti-APC

MicroBeadsFcR

Blocking培养:NH

Expansion

Medium

(2x500

mL)CytoMix

-

CD271

(LNGFR)质量控制:ntroductory

offerNH

AdipoDiff

Medium

(12x8

mL)NH

OsteoDiff

Medium

(12x5

mL)Ads

in

Blood

(April

&

Mai)MSC

Research

Tool

Box

-

CD271

(LNGFR)人类MSC的标准化研究CD

271又叫做低亲和力神经生长因子受体(LNGFR),是迄今为止发现的最佳MSC分选标志，可用于直接从人类骨髓中分选MSC。分选后CD

271(LNGFR)+MSCs是一群均一的MSC细胞群，而CD

271阴性细胞成分中没有成纤维细胞克隆形成单位(CFU-F)活性。与贴壁培养的MSC相比，CD

271分选的MSC增殖能力增强10－1000倍。CD

271分选和扩增的MSC保留了多系分化潜能，具有分化为脂肪细胞和成骨细胞、软骨细胞、内皮细胞的可塑性。cumulative

population

doubling

(CPD)Ads

in

Blood

(April

&

Mai)MSC

Research

Tool

Box

-

CD271

(LNGFR)人类MSC的标准化研究Nadia

Quirici等，Experimental

Hematology

30(2002)783–791MACS小鼠干细胞分选试剂盒：Lineage

Cell

DepletionKit:去除成熟和分化的造血细胞，预富集造血干祖细胞。CD117

Micro

Beads：分选早期和晚期干祖细胞。Anti-Sca-1

Micro

Beads：分选造血干细胞和早期造血祖细胞。CD117+Sca-1+lin–细胞也有非造血分化潜能，如分化为肌细胞、内皮细胞、肝细胞、神经系统细胞。CD45/Ter119微珠去除分选骨髓细胞，可以得到MAPCs,也有体外分化为多种细胞的潜能。小鼠骨髓造血干细胞(HSC)定义为Sca-1+、

CD117+(c-kit)、Thy-1low和lin-细胞.HEA:human

epithelial

antigen(melanoma

chondroitin

sulfate

proteoglycan

(MCSP)+MACS®技术分选人类肿瘤细胞New上皮肿瘤细胞根据表面抗原进行分选与分析：

Anti-HEA

(Ep

CAM)

Micro

BeadsAnti-Erb

B-2(HER-2/neu)Micro

Beads根据细胞内细胞角蛋白分选与分析：cytokeratin黑色素瘤细胞分选：

Anti-melanoma

MicroBeads其它非造血系统肿瘤细胞:

CD45

MicroBeads去除白细胞骨髓瘤细胞：

CD138

MicroBeads霍奇金氏淋巴瘤/RS细胞：

CD30

MicroBeadsM

D

0

0

4

4

.

0

2Cytokine

Secretion

Assay

is

a

completelynew

technology

(gota

different

colour

on

the

slide).

Analysis

and

also

enrichment

of

extremely

rare

antigen-specificTcells,

for

example.MACS®技术分选凋亡细胞New刺激、分化、凋亡、脱颗粒、坏死PS暴露M

D

0

0

4

4

.

0

2PS暴露细胞被Annexin

V微珠磁性标记磁性标记细胞滞留在分选柱上PS暴露细胞被洗脱下来Cytokine

Secretion

Assay

is

a

completelynew

technology

(gota

different

colour

on

the

slide).

Analysis

and

also

enrichment

of

extremely

rare

antigen-specificTcells,

for

example.MACS®技术分选转染细胞New应用于功能基因、信号转导研究、药物筛选和标记技术。M

D

0

0

4

4