autoMACS™ Pro Separator – 细胞自动分选的标准

1. autoMACS™ Pro Separator –细胞自动分选的标准

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3. 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

4. 基于MACS®技术 • Automated, sensor-controlled multisample labeling with MACS® MicroBeads and subsequent cell sorting :

5. 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

6. 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

7. 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..........

8. 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

9. 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

10. autoMACSTM Pro Separator – 主要部件

11. 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

12. 主要部件(I) • 触摸屏 • 机械手 • 条码阅读器 • 试剂瓶支架 (4) • 试管架探测 1 2 3 4 4 5

13. 主要部件(II) • 机械手 • 采样针/ 阳性组分出口 • 阴性组分出口 • MiniSampler固定接口 • 存储卡插槽 • 电源开关 5 1 2 3 6 4

14. 主要部件(III) – MACS® MiniSampler • 低温试管架位置 • 试剂架 • MiniSampler 插口 • MiniSampler 保护盖插口 • the autoMACSTM Pro 背后插头 2 1 3 4 5 • Move Racks in x-direction (left-right) • Direct connection to the instrument • Lid for sample protection

15. 低温试管架 • Cooling of sample and sorted fractions to sustain cell viability • MiniSampler lid for sample protection • Automatic recognition of different tube racks • Cooling Tube Racks • Reagent Rack 4 • Holds up to 4 vials for automated sample labeling

16. MACS® 缓冲液 autoMACS® Pro • Connects directly to the instrument • Controlled for cell separation performance (USP) • High-quality ingredients • Three Solutions are used to operate and maintain autoMACS™ Instruments * 70% Ethanol prepared from absolute (100%) Ethanol

17. 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

18. 如何使用这台细胞分选仪器?

19. 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。

20. 如何使用autoMACS pro? 4. Prime autoMACSTM Pro Separator 1. Choose a cell separation approach 5. Program a separation template (up to six samples) 2. Prepare cell Sample 3. (optional) manual labeling of cell samples 6. Run autoMACSTM Pro Cell Separation 7. Run “Sleep” or “Store” program for storage

21. 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

22. 选择分选程序 Eliminate cell population(s), obtain 'untouched' cells Select cell populations according to particular cell surface antigen Goal Strategy Program Positive selectionMagnetic labeling of target cells Depletion Magnetic labeling of cells other than the target cells Normal to high frequency cells Rare cells, or purity increase Normal to high antigen expression Low antigen expression POSSELPositive selection POSSELDDouble positive selection DEPLETEDepletion POSSEL_SSensitive positive selection POSSELD2Double positive selection DEPLETESSensitive depletion POSSELDSSensitive double positive selection DEPL05 and DEPL025 Special sensitive depletions * automatic Dilution 1:3 prior to uptake ** Stage loading (up to 7 mL), not applicable to autolabelling A_Depl/A_Depls** Large scale depletion POSSELWB* Blood, marrow

23. 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

24. 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

25. 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 Status Log list Option

26. "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

27. "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

28. "Status": Sensor-controlled processing • Monitoring Incubation, Processing, Washing,... • Labeling is scheduled to allow optimal processing times • Separation / Wash is performed accordingto positions • Bottle illumination supports monitoring fromdistance

29. "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"

30. "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

31. Bottle illumination displays instrument status

32. Volumes Software 1.5.xx Separation only Maximum volume per sample 2.5 mL 12.5 mL 50 mL

33. Volumes Software 2.0.1.5& Separation Autolabelling Chill 5 Whole Blood Separationonly Chill 15 Whole Blood Separationonly Chill50 Whole Blood Autolabelling

34. Volumes Software 2.0.3.4& Separation Autolabelling Chill5 Whole Blood Autolabelling Chill15 Whole Blood Autolabelling Chill50 Whole Blood Autolabelling

35. 演示准备

36. 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 BarcodesCustomer: Prepare 70% Ethanol (from 100%)

37. 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

38. 安装 • 将机器放置在牢固无振动的桌面上 • 连接MACS® MiniSampler • 连接主电源线, fluid sensor-, barcode reader- , and MiniSampler cords • 放入缓冲液，检查空的废液瓶 • 打开autoMACSTM Pro Separator • 可选：安装新的分选柱

39. autoMACS™ Pro fluid containers • 连接液体传感器 • 按照说明准备缓冲液

40. Results, Examples • From abundant to rare cells

41. CD8 microbeads，human

42. 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

43. CD4 MicroBeads, human PBMC; purity: > 98% Program: hMB.CD4 (autolabeling) / Possel / Qrinse

44. CD4 MicroBeads, human % CD4 cells in negative fraction Start percentage CD4 autoMACS™ program CD4 Purity time 39,3% 39,3% 39,3% 39,3% 97,9% 94,7% 99,6% 97,2% POSSEL POSSEL_S POSSELD POSSELDS 2.5 min 5 min 7 min 8.5 min 16,7% 3,2% 17,3% 2,21% CD4-Cy5 stained sample Results analysed by flow cytometry By Courtesy of A. Sheffold, A. Thiel - Berlin, Germany

45. Whole Blood CD14 MicroBeads, human Anticoagulated whole blood; Purity: > 98% manual labelling, program Posselwb /Rinse

46. 阳性分选CD34+细胞CD34 MicroBead Kit, human • 原始标本 • 0.37% • CD34+ cells • autoMACS Separator, • by Courtesy of • Dr. Haberstock, • Dr. Allam, Hautklinik Bonn, Germany • 7-AAD • Count • CD34-PE • CD34-PE • 富集组分 • 95.38% • CD34+ cells • 来源: 人脐带血; autoMACS 程序POSSELD • 7-AAD • Count • CD34-PE • CD34-PE

47. Example: Multiple cell sorting strategy • NK cell subsets

48. CD56+CD8+/CD8- NK Cell Isolation Kit PureNK-subset cells Pre-enrichment NK cells PBMCs Labeling of unwanted cells with the Biotin-Antibody Cocktail and Anti-Biotin MicroBeads Depletion of unwanted cells Magnetic labeling using subset-specific MicroBeads Magnetic Separation: CD8+: possel -> Flow through CD8- : Depl05 Elution of CD8+target cells

49. CD56+CD8+/CD8- NK Cell subsets CD56+CD8+ NK cells Pre-enrichedNK cells PBMCs CD56+CD8- NK cells CD56 CD8

50. 应用领域 多发性骨髓瘤 嵌合体 诊断实验室 免疫学实验室 WB CD34 即将面世! 干细胞实验室 流式核心机构 • 神经实验室 代替 补充