autoMACS™ Pro Separator –
细胞自动分选的标准
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
中心已对本次培训讲座录制视频，需要观看视频
的老师和同学请带移动硬盘到科技楼北810房（
原北806房）拷贝视频。
科研仪器管理中心
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
autoMACS™ Pro Separator – 内容
 特点
 主要部件
 如何使用?
 安装 & 演示
 维护 & 部件
 总结
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
基于MACS® 技术
 Automated, sensor-controlled multisample labeling with
MACS® MicroBeads and subsequent cell sorting
:
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
autoMACS™ (Pro) 细胞分选
 基于MACS® 技术
 自动的多样品处理
 简单快捷
 标准化的程序, 重复性好可靠的
结果
 成本低，分选柱可以使用高达
100次
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
MACS® 技术 – 优点
 MACS® 分选柱技术
 温和：细胞活性高，功能活跃
 极佳的纯度、得率和活性
 灵活的分选策略:
– 阳选 – 去除 – 多参数分选
 分选任何细胞: 数百种种分选试剂+间标微珠
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
MACS® 技术 – 优点
 MACS® 微珠
 超顺磁性纳米颗粒, 偶联特异性抗体
 无毒性, 可生物降解：标记的细胞可直接进行下游试验，如流式分析，
细胞培养，WB, PCR, 质谱分析等等..........
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
autoMACS™ (Pro) 试剂兼容性
 直标微珠
(for human, non-human primate, mouse, rat)
 全血微珠
 间标微珠(Anti-Fluorochrome,
Anti-Biotin / Streptavidin / Anti-Immunglobulin)
 MicroBead Kits, Cell Isolation Kits
 2-step Cell Isolation Kits, MultiSort Kits*
 MACSelect Transfected Cell Selection
 Cytokine Secretion Assay - Enrichment Kit*
* Possibly not fully automated
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
autoMACS™ 技术 – 主要优势
 基于MACS® 技术，使用 50nm MACS® 微珠
 经过时间验证的品质：1999 年首次推出
(“classic“ autoMACSTM+autoMACS ProTM=3000 instruments since 2008)
 细胞自动分选的先锋, 上千篇文献支持
 简便快捷
 操作者所需的培训最少, 降低了实验室的工作量
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
autoMACSTM Pro Separator – 主要部件
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
自动多样本分选
 MACS® MiniSampler & 机械臂
 自动标记(可选)
 低温试管架
for 5, 15, 50mL sample tubes
 传感器控制的分选过程
 12个分选程序
 全自动传感器控制的上样和洗脱
 样品之间强制性自动清洗
 缓冲液有传感器控制
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
主要部件(I)
1. 触摸屏
1
2. 机械手
3. 条码阅读器
2
4. 试剂瓶支架 (4)
5. 试管架探测
3
4
4
5
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
主要部件(II)
1. 机械手
5
2. 采样针/ 阳性组分出口
3. 阴性组分出口
1
2
4. MiniSampler固定接口
5. 存储卡插槽
3
6. 电源开关
6
4
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
主要部件(III) – MACS® MiniSampler
1. 低温试管架位置
2
2. 试剂架
1
3
3. MiniSampler 插口
4
4. MiniSampler 保护盖插口
5
5. the autoMACSTM Pro 背后插头



Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
Move Racks in xdirection (left-right)
Direct connection to
the instrument
Lid for sample
protection
2010/12
低温试管架
 Cooling Tube Racks
Rack
type
Max. no.
of tubes
Max. vol.
per sample
Max. no.
of cells
Chill 5
6 (5 mL)
2.5 mL
5×108
Chill 15
5 (15 mL)
12.5 mL
2.5×109
Chill 50
3 (50 mL)
50 mL
4×109
 Reagent Rack 4
 Holds up to 4 vials for
automated sample labeling
Basic Instrument Training – autoMACS™ Pro Separator
 Cooling of sample and
sorted fractions to sustain
cell viability
 MiniSampler lid for sample
protection
 Automatic recognition of
different tube racks
Internal use only!
2010/12
MACS® 缓冲液 autoMACS® Pro
 Connects directly to the instrument
 Controlled for cell separation
performance (USP)
 High-quality ingredients
Buffer
Color-code
Order no.
Running Buffer
blue
130-091-221
Washing Solution
green
130-091-222
Storage Solution*
black
n.a.
 Three Solutions are used to
operate and maintain
autoMACS™ Instruments
* 70% Ethanol prepared from absolute (100%) Ethanol
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
可重复使用的分选柱
 14 天, 或 100 次分选
 特殊的, 对细胞温和的基质
 设计: 不会暴露在空气中
 高性能 – 细胞分选在几分钟之内
完成
 105 up to 4 x 109 总细胞（有核细胞）
 up to 2 x 108 磁性标记的细胞
 up to 15 mL 全血
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
如何使用这台细胞分选仪器?
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
Before you start
 准备单细胞悬液
 避免细胞团块
 避免过量的死细胞
 严格按照说明书的说明进行磁珠标记
 手动和自动分选，磁珠标记的方法是相同的。
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
如何使用autoMACS pro?
4. Prime
autoMACSTM Pro
Separator
1. Choose a cell
separation approach
2. Prepare cell
Sample
3. (optional)
manual labeling
of cell samples
Basic Instrument Training – autoMACS™ Pro Separator
5. Program a
separation template
(up to six samples)
6. Run autoMACSTM
Pro Cell Separation
Internal use only!
7. Run “Sleep”
or “Store”
program for
storage
2010/12
磁珠标记
手动标记 vs. 自动标记
 兼容试剂盒 :
 Standard Cell Isolation Kits
 Standard MicroBeads
(Cell frequencies higher than 5%)
 Autolabeling volumes 0.2 – 8 ml
 (Whole Blood MicroBeads: 0.25 - 8 mL)
 Limitations (determine customer priority):
 Direct MicroBeads for rare cells (Cell frequencies less than 5%)
 Kits containing wash / centrifugation steps between labeling steps
e.g. 2-step kits, Indirect MicroBeads
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
选择分选程序
Goal
Strategy
Program
Select cell populations according
to particular cell surface antigen
Eliminate cell population(s),
obtain 'untouched' cells
Positive selection
Depletion
Magnetic labeling of target cells
Magnetic labeling of cells
other than the target cells
Normal to high
frequency cells
Rare cells, or
purity increase
Normal to
high antigen
expression
POSSEL
POSSELD
DEPLETE
Positive
selection
Double positive
selection
Depletion
Low antigen
expression
POSSEL_S
POSSELD2
DEPLETES
Sensitive
Double positive
positive selection selection
* automatic Dilution 1:3 prior to uptake
** Stage loading (up to 7 mL), not
applicable to autolabelling
Basic Instrument Training – autoMACS™ Pro Separator
Sensitive depletion
POSSELDS
DEPL05 and DEPL025
Sensitive double
positive selection
Special sensitive depletions
POSSELWB*
A_Depl/A_Depls**
Blood, marrow
Large scale depletion
Internal use only!
2010/12
Priming, Rinsing, Cleaning, and Sleep
 Priming
 Program "Rinse" from Menu "Wash only"
 Change from Ethanol-filling to Running buffer
 QRinse (Quick Rinse)
 2 min, rinse and refill with Running buffer
 Standard short rinse, abundant cells
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
Priming, Rinsing, Cleaning, and Sleep II
 Rinse
 5 min, rinse with Washing solution; rinse and refill with Running buffer
 Mandatory after sticky samples, e.g. whole blood or tissues
 Recommended before rare cell isolations
 Sleep
 5 min, rinse with Washing solution; rinse and refill with 70% Ethanol
 prepare instrument for O/N or weekend storage (up to 4 days)
 mandatory daily maintenance
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
operation with intuitive software
 Reagent menu—load protocol information by scanning reagents
 Separation menu—program separation sequence and start process
 Status menu—monitor instrument status and progress of separation
 Log list menu—access process details and history
 Option menu—access special programs and user settings
Reagent
Separation
Basic Instrument Training – autoMACS™ Pro Separator
Status
Log list
Internal use only!
Option
2010/12
"Reagent": Load up to 4 MACS® Reagents
 Note reagent positions (R1-R4)
 Activate barcode reader by pressing a button
 Scanned reagents are displayed as 8-digit code
 Product information is displayed on info screen
 Templates can be saved / reloaded
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
"Separation": Program a separation template
 Displays loaded MACS® reagents in Menu
"Separation - Labeling"
 Pre-selected separation and wash programs
 Define sample volumes
 Side-by-side processing of manual and
automated labeled samples possible
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
"Status": Sensor-controlled processing
 Monitoring Incubation, Processing,
Washing,...
 Labeling is scheduled
to allow optimal processing times
 Separation / Wash is performed according
to positions
 Bottle illumination supports monitoring from distance
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
"Log list": All actions are saved
 All actions are logged by date and time
 Status monitoring is done by symbols
 For more information on actions select "Details"
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
"Option": Maintenance & user settings
 "Col_ex" program for column exchange
(displayed in "Status" menu)
 Access to program "Safe"
(maintenance procedure)
 Access to program "Store" (long-term storage,
installation of column substitutes
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
Bottle illumination displays instrument status
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
Volumes Software 1.5.xx
Separation only
Maximum
volume per
sample
2.5 mL
12.5 mL
50 mL
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
Volumes Software 2.0.1.5
Separation & Autolabelling
Chill 5 Whole Blood Separation only
Chill 15 Whole Blood Separation only
Chill50 Whole Blood Autolabelling
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
Volumes Software 2.0.3.4
Separation & Autolabelling
Chill5 Whole Blood Autolabelling
Chill15 Whole Blood Autolabelling
Chill50 Whole Blood Autolabelling
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
演示准备
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
autoMACS™ Pro Separator
准备安装或演示
–细胞数
总体积与起始细胞总数
– 应用
Positive Isolation / Untouched Isolation / Depletion
Downstream Applications: Purity - Recovery
– 试剂
Order Regular Reagents (not samples)
Confirm Barcodes
Customer: Prepare 70% Ethanol (from 100%)
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
体积 & 细胞数量
 Ensure that enough cells are
provided
 Autolabeling volumes:
0.2 - 8.0mL
 Standard MicroBeads
– Min 2x107 cells, for less cells use same volume
– Max 6.5x108 cells
 Cell Isolation Kits
– Min 4x107 cells, for less cells use same volume
– Max 6.5x108 cells
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
安装
1.
将机器放置在牢固无振动的桌面上
2.
连接MACS® MiniSampler
3.
连接主电源线, fluid sensor-, barcode reader- , and MiniSampler
cords
4.
放入缓冲液，检查空的废液瓶
5.
打开autoMACSTM Pro Separator
6.
可选：安装新的分选柱
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
autoMACS™ Pro fluid containers
 连接液体传感器
 按照说明准备缓冲液
Container
Content
Color-code
Order no.
autoMACS™
Running Buffer
PBS, 0.5% BSA, EDTA
Blue
130-091-221
autoMACS™ Pro
Washing Solution
Salt solution
with detergent
Green
130-092-987
Storage Solution
(70% ethanol)
100% ethanol,
analytical grade
Black
N/A
Waste
Empty
Red
N/A
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
Symbol
2010/12
Results, Examples
From abundant to rare cells
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
CD8 microbeads，human
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
CD8 microbeads，human
验收结果。Possel程
序分选。分选后阳性部
分淋巴细胞门内CD8+T
细胞比例99.6%。阴性
组分CD8+T细胞0.96%。
回收率93%。Flowjo数
据分析。
分选操作：潘涛
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
CD4 MicroBeads, human
PBMC; purity: > 98%
Program: hMB.CD4 (autolabeling) / Possel / Qrinse
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
CD4 MicroBeads, human
autoMACS™
program
Start
percentage
CD4
CD4
Purity
% CD4
cells in
negative
fraction
POSSEL
39,3%
97,9%
16,7%
2.5 min
POSSEL_S
39,3%
94,7%
3,2%
5 min
POSSELD
39,3%
99,6%
17,3%
7 min
POSSELDS
39,3%
97,2%
2,21%
8.5 min
time
CD4-Cy5 stained sample
Results analysed by flow cytometry
By Courtesy of A. Sheffold, A. Thiel - Berlin, Germany
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
Whole Blood CD14 MicroBeads, human
Anticoagulated whole blood; Purity: > 98%
manual labelling, program Posselwb /Rinse
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
阳性分选 CD34+ 细胞
CD34 MicroBead Kit, human
原始标本
0.37%
CD34+
Count
7-AAD
autoMACS Separator,
by Courtesy of
cells
Dr. Haberstock,
CD34-PE
CD34-PE
富集组分
Count
7-AAD
95.38%
Dr. Allam, Hautklinik
Bonn, Germany
CD34+
来源: 人脐带血;
cells
autoMACS 程序
POSSELD
CD34-PE
Basic Instrument Training – autoMACS™ Pro Separator
CD34-PE
Internal use only!
2010/12
Example: Multiple cell sorting strategy
 NK cell subsets
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
CD56+CD8+/CD8- NK Cell Isolation Kit
PBMCs
Labeling of unwanted
Depletion of
cells with the Biotinunwanted cells
Antibody Cocktail and
Anti-Biotin MicroBeads
Pre-enrichment
NK cells
Magnetic
labeling using
subset-specific
MicroBeads
Magnetic
Separation:
CD8+: possel
Pure NKsubset cells
Elution of CD8+
target cells
-> Flow through
CD8- : Depl05
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
CD56+CD8+/CD8- NK Cell subsets
PBMCs
CD56+CD8+
NK cells
Pre-enriched
NK cells
CD56
CD56+CD8NK cells
CD8
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
应用领域
诊断实验室
多发性骨髓瘤
嵌合体
免疫学实验室
流式核心机构
 神经实验室
代替
补充
WB CD34
干细胞实验室
即将面世!
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
全血应用
autoMACS Pro 结合全血磁珠:
 快速轻松的分选淋巴细胞亚群
 一次处理多个样品
 标准化
 自动化 – 与液体工作站组合使用（可选）
 目标市场的机会有很大的潜力和很好的参考文献：
–临床医学研究：嵌合体研究
–多发性骨髓瘤检测(诊断应用)
参考文献
30分钟以内从全血中得到纯度大于 90% 的单核细胞
样品在4°C保存和处理, 对基因表达的影响最小化
样品在室温保存和处理, 对基因表达有强烈影响
分选细胞的RNA基因表达谱分析显示极低的背景，导致更高的相关系数
参考文献 – 竞争优势
竞争优势
EasySep 和RoboSep 在室温进行操作并且需要红细胞裂解.
The autoMACS® Pro 可以把细胞维持在4C并且不需要对血样进行预处理。根据海报的数据，使用MACS® 技
术在低温下处理细胞，基因表达谱的分析数据更加理想，尤其是处理多人样本的时候
参考文献 – Clinical SOP
结论:
 使用MACS 技术建立了直接从全血中自动分选CD14+单核细胞的SOPs.
 这些方法手动处理最少, 只需30分钟, 可以得到充足的产率，和质量极佳的RNA可用于基因表达谱
分析。
 表达谱分析表明了手动和自动分选之间的高度一致性
 与全血或PBMCs相比，经过富集的CD14+ 的单核细胞可以提高转录组检测的灵敏度。
MACS® 细胞因子分泌细胞分选
捕获试剂
分泌的
细胞因子
抗-PE 微珠
检测抗体
分泌细胞
因子的细胞
•
抗原特异性再刺激
•
捕获试剂标记
•
分泌阶段
•
(可省略) 磁性标记
Cohen, G.B. (2002)
Virology 304: 474–484.
 “由于我们的流式细胞仪不进行HIV感染性样品的分选所以这些细胞没有使用FACS技术分
选”。作者也指出“没有使用细胞内染色后流式分选IFN-γ分泌细胞的方法分选分泌INF-γ的
细胞是因为细胞内染色之前需要使用多聚甲醛对细胞进行固定，固定会降低细胞内RNA含
量，使得使用RNA进行PT-PCR检测十分困难。相比之下，IFNγ抓捕技术分选出的细胞是
活性细胞，其RNA完好无损，适合做RNA提取和后续的RT-PCR检测”
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
维护 & 配件
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
维护
 日常维护
 Priming: Program "Rinse"
 Shut down: Program "Sleep" for overnight storage (fill with Ethanol)
 周期性维护
 更换分选柱 (Program “Option - Special - Col_ex”): 14 天 或者 100 分选后,
那个最先实现。（这点很重要，生锈的铁颗粒会损伤机器！）
 清洁柱塞泵: 1-2 month
 长期不用: Program “Store” (替换柱子)
 清洁管路(同样可以用于去除污染): Program "Safe", cleaning procedure
with hypochlorite – MACS® Bleach solution
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
总结
 无需守护，可同时处理六个样本
 进样,洗脱, 系统清洗完全自动化
 样本低温
 分选过程安全可控：传感器控制的仪器
 基于著名的MACS® 技术
 紧凑的台式设计，可放于生物安全柜
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12
MACS劣势
不能根据细胞大小分选细胞
不能分选在胞内表达某种蛋白的活细胞（GFP等）
三个参数以上的顺序阳性选择（操作繁琐耗时）
能分选阳性和阴性细胞不能分选(low, mid, high)细胞群
MACS+FACS
Integrated Solutions for research
Basic Instrument Training – autoMACS™ Pro Separator
Internal use only!
2010/12