Magnetic Cell Separation

Magnetic Cell Separation
with AutoMACS(Pro) 정 금 희

Cell separation with MACS® Technology
Higher organisms such as man bears many different cell types. Here, different blood cell types are shown, all develping from a central stem cell. For a lot of applications it is necessary to obtain one single cell type. MD

Magnet Activated Cell Sorting (MACS)
• 1988년 독일 쾰른 대학 유전학 연구실 Stefan Miltenyi 고안 • 원리 : 세포의 표면 항원 혹은 분자적 특징을 이용하여 bead를 붙이고 자석과 column으로 세포나 분자 물질들을 분리 • 구성 : Microbead, Column, 자석

MACS Microbead • Non - toxic • Straight to experiment or cell culture
• 구성 물질 : Iron Oxide, Polysaccharide • 크기 : 직경 30 ± 20 nm • Stable colloidal suspension • Biodegradable • Non - toxic • Don’t activate cells, influence function and viability • Straight to experiment or cell culture

Compatible with flow cytometry
• No bead detachment required • Only 20-30% of binding sites occupied • MACS® separation + flow sorting • Decreased flow sorting time ad 1. MicroBeads do not affect the scatter properties of the cells, you may go straight from separation to analysis or to experiment ad 2. Very few material allows parallel staining with fluorochrome-conjugated antibodies. ad 3. MACS can be used as a pre-enrichment for flow sorting (especially the autoMACS) in order to get rid of the main part of the cells. MD

CD8+ T cells isolated by MACS® Technology
To see the MicroBeads you have to make a cross-section through the cell and perform a transmission electron micrograph. The MicroBeads are sitting on the surface (arrows). You can also see the douple membrane of the cell. MD

Magnetic field generated in an MS Column
Reason for the steel balls: They generate a high gradient magnetic field with extremely strong magnetic forces in the „edges“. That allows Miltenyi Biotecto put extremely few material on the cell surface, i.e. the tiny MicroBeads. MD

Choose a cell separation approach
• Positive selection or Depletion • One parameter sorting or Multi-sort • Direct or Indirect Microbead

Positive selection strategy
Magnetic labeling with MACS® MicroBeads Elution of positively selected cells Flow through with unlabeled cells

Negative selection strategy (depletion)
Target Cells Negative Fraction The 2nd MACS separation strategy is the Depletion Method. It is also known as the Untouched method. - First the cells are incubated with MACS MicroBeads. Instead of target cells being magnetically labeled, all cells other than the target cell is magnetically labeled. This is achieved by have a MB cocktail that recognizes all the other non-target cells present within the sample. - The sample is passed through magnetized column and all magnetically-labeled non-target cells are retained in the column while the target cells flow into the collection tube. This unlabeled target cell fraction is now called the “Negative Fraction”.

Isolation of untouched cells
Subsets(1) : Depletion followed by positive selection Magnetic labeling of unwanted cells Isolation of untouched cells Laleling of target cells Elution of target cells

Subsets(2) : Multi-sort strategy
• Positive selection이나 depletion 방법으로 세포를 얻어내고, 이들 중 또 다른 특정 항원이 발현된 세포를 분리하는 방법 • 목적 항원을 여러개 가진 세포 분리에 적합

Direct Bead • Microbead가 붙어있는 일차 항체와 세포를 일정 시간 동안 반응 후 이를 분리
• 시간/노력 절감 • 세포 손실 적음 • Low Background

Indirect Bead • 세포에 일차 항체 (Purified, Biotinylated,
FITC/PE-conjugated…)를 반응시킨 후, 적합한 이차 항체-bead를 적용 • Direct용 항체-bead가 없는 경우 • 실험자가 이미 일차 항체를 가지고 있는 경우 • 항원의 발현율이 낮은 경우

Direct Magnetic Labeling (Eg. CD4, CD8)
Indirect Magnetic Labeling Anti-Fluorochrome MicroBead Fluorochrome-Tagged Primary antibody Biotin-tagged Primary antibody Anti-Biotin / Streptavidin MicroBead There are 2 kinds of labeling methods for positive selection depending on the availability of a MACS MB. - If Miltenyi have the MB that directly recognise the target cells of the customer, the customer can simply use the Direct magnetic labeling method for the isolation of their cells. Eg are CD4 MB, CD8MB - However If a customer is interested in isolating a particular MicroBeads but Miltenyi does not have the direct MicroBeads, cells purification can still be performed using the indirect Magnetic Labeling strategy. All the customer need is to find an antibody that targets the cell-of-interest. Purification can then be performed using our Anti-Fluorochrome MB, Anti-Biotin MB or MB against the species of the Primary ab. Untagged Primary antibody Anti-Primary Antibody Species MicroBeads

Materials & Methods * 필요한 시약 1> 항체 - 세포 분리용 : Microbead가 결합되어있는 항체
- 세포 확인용 : 형광이 결합되어 있는 항체 2> Buffer - Degassed PBS (pH7.2)/ 2mM EDTA and 0.5 % BSA 실험방법 1> 세포, buffer, column 준비 2> 세포에 항체-microbead 처리 3> Column에 sample loading 4> 수세 (non-specific bound cell on column) 5> Column을 자석에서 떼어낸 후 buffer로 elution 6> Flow cytometer를 이용하여 확인

Result Analysis • Purity : Flowcytometer를 이용하여
Original, Positive, Negative fraction의 purity 측정 • Recovery : 분리 전 / 후의 세포수의 비교 • Viability : Trypan-blue, PI, DAPI 등의 dye를 사용

Manual Devices MiniMACS MidiMACS VarioMACS SuperMACS

autoMACS™ Magnetic Cell Separation
Renowned First automated magnetic cell separator Introduced in 1999 Over 1450 instruments world-wide Time-effective, fast, and easy sample separation Standardized procedure, reproducible results Only minimal operator training required Lower workload for busy labs

autoMACS™ Pro Separator
NEW Now, high quality cell separations are even easier!

autoMACS™ Pro Separator Walk-away cell sorting of multiple samples
Processing of up to six samples MACS® MiniSampler, robotic arm and MACS® Cooling Tube Racks Automation Sample resuspension, uptake, elution Rinsing of tubes, columns, uptake port, and elution port Intuitive software, operated by touchscreen 10 pre-set separation programs, 2 rinsing programs Optimized sensor-controlled pro-cess Sample uptake Buffer supply, color-coded Column status Sampler and rack detection

autoMACS™ Pro Separator Easy operation
Intuitive software Touchscreen Pre-set separation and rinsing programs Monitoring of buffer and column status, sampler, and cooling tube racks Ready-to-use buffers autoMACS™ Running Buffer autoMACS™ Pro Washing Solution Buffer bottle illumination Reusable autoMACS™ Columns Compact benchtop design Fits in laminar hood

autoMACS™ Pro Separator Key components (I)
Touchscreen Washing station Robotic arm Rack detection Fluid container (4 x) 1 3 5 2 4

autoMACS™ Pro Separator Key components (III)
1. Separation column 1 2. Separation column 2 Pump Five valves 4 3 1 2 4

autoMACS™ Pro Separator MACS® Cooling Tube Racks for optional cooling
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 Optional cooling to sustain cell viability Lid for maximum sample protection Automatic recognition of different tube racks

autoMACS™ Pro Separator How to work with the instrument
1. Choose a cell separation approach 4. Prime autoMACS Pro Separator 5. Define a separation sequence (up to six samples) 2. Prepare cell sample 6. Perform autoMACS Pro Cell Separation 3. Label cells magnetically 7. Run “Sleep” or “Store” program for storage

autoMACS™ Pro Operation Fluid containers
Connect fluid sensor cables Fill or empty fluid containers accordingly Container Content Color-code Order no. Symbol autoMACS™ Running Buffer PBS, 0.5% BSA, EDTA Blue autoMACS™ Pro Washing Solution Salt solution with detergent Green Storage Solution (70% ethanol) 100% ethanol, analytical reagent grade, diluted with distilled H2O Black NA Waste Empty Red

autoMACS™ Pro Operation Fluid containers
Bottle illumination indicates status of the instrument Code Status User action Green Ready for separation No action required Blue Instrument operating Yellow Not ready for separation Run wash program (“Rinse” or “Qrinse”) Red Error Check screen for error correction Purple Program “Sleep” is completed Switch off autoMACS™ Pro Separator Blinking Action required Check screen for required action

autoMACS™ Pro Operation Software
Bottle and fluid level status Column status Tube rack recognition MiniSampler recognition Separation sequence: White: finished programs Orange: in operation Yellow: wait position 2 1 3 4 5

autoMACS™ Pro Operation Choose a separation program
Positive selection versus depletion Single-column versus double-column selection Standard mode versus sensitive mode Goal Strategy Program Obtain a cell population expressing a particular cell surface antigen Eliminate cell subpopulation(s) from your cell sample and obtain “untouched” cells Positive selection Magnetically label the target cells Depletion Magnetically label cells other than the target cells Cells with normal to high frequency Rare cells, or increase of purity Normal to high antigen expression Low antigen expression POSSEL Positive selection POSSELD Double positive selection DEPLETE Depletion POSSEL_S Sensitive positive selection POSSELD2 Also for small blood volumes DEPLETES Sensitive depletion POSSELWB For blood volumes from 3 mL to 15 mL DEPL05/ Special depletion NEW ! POSSELDS Sensitive double- positive selection

autoMACS™ Pro Operation Rinsing and cleaning programs
Qrinse (Quick Rinse; standard short rinse) 1.5 min Rinsing and refilling with autoMACS™ Running Buffer Rinse 4 min Rinsing with autoMACS™ Pro Washing Solution; rinsing and refilling with autoMACS™ Running Buffer For priming of the autoMACS™ Pro Separator Recommended before isolation of rare cells and between whole blood separations

autoMACS™ Pro Maintenance Programs and other
Sleep Rinsing with autoMACS™ Pro Washing Solution; rinsing and refilling with Storage Solution Before instrument is switched off for overnight storage Safe Recommended for decontamination with hypochlorite solution Store Prior to storage period longer than 2 weeks Substitute columns should be installed Column exchange Every 2 weeks Other Cleaning the surface of the instrument: as necessary Cleaning of pump syringe: every 1–3 months Pump seal or pump syringe replacement: once per year Valve replacement: once per year

Standardized procedure for reliable results Examples
Separation results with autoMACS™ Magnetic Cell Sorting Positive selection and depletion From rare to frequent cells From PBMCs, whole blood, tissue From any species

MACS® Whole Blood MicroBeads
Developed for autoMACS™ and autoMACS Pro Magnetic Cell Sorting Human hematopoietic subsets directly from whole blood Rapid—no density gradient centrifugation, no erythrocyte lysis Ideal for molecular studies, e.g., chimerism analysis Safe handling of hazardous samples, e.g., HIV samples CD15, CD3, CD4, CD8, CD56, CD19, CD45, CD14 Whole Blood MicroBeads autoMACS™ Separator: small sample volumes from 0.25–3 mL autoMACS™ Pro Separator: sample volumes from 0.25–15 mL

Positive selection from whole blood Monocyte isolation with Whole Blood CD14 MicroBeads
Source: Anti-coagulated whole blood Program: Posseld2 Purity: >98%

Mouse dendritic cells from spleen CD11c MicroBeads, mouse
Source: mouse spleen Program: Posseld Purity: >93%

Untouched isolation of mouse CD4+ cells CD4+ T Cell Isolation Kit, mouse
Source: mouse spleen Program: Deplete Purity: >90%

AutoMACS vs Flow Cytometry
Fluorescence activated cell sorting Flow sorting time: cells/sec = 6 x 105 cells/min = 3.6 x 107 cells/hr FACSAria (BD Biosciences): cells/sec Flow Cytometry autoMACS Separator 107 cells min cells 5min 108 cells 2hr 40min cells 5min 109 cells 27hr cells 5min Flow cytometry is a common immunological method that researchers use for analysing cells within the sample and also for sorting out their cell-of-interest using fluorochrome-conjugated antibodies. The principle of flow cytometry sorting is similar to flow cytometry analysis in that laser is used for the analysis of the expression profile of each cell. Cells that express the fluorochrome-conjugated antibody on its surface will be identified and transferred to the collection tube when it pass through the laser. The time necessary for flow cytometry sorting depends on the total cell count and the type of flow cytometry machine that the researcher have. On average each cytometer can process 10k cells/ sec. The fastest cytometer is the BD FACSAria which can process up to 50k cells/sec. How does this compare with our automated autoMACS machine? For 10e7 cells, flow cytometer takes 16 min to sort while the autoMACS takes only 5min. The difference might not be much. But look on... For 10e8 cells, flow cytometer now need 2hr 40min to sort while autoMACS still only require 5min What about 10e9? It taks a whooping 27hr for flow cytometry! And autoMACS still only require 5min

AutoMACS vs Flow Cytometry
How does flow cytometry

소모품 (Columns) 단가 비교 규 격 Re-use 단가/Test MS LS XS Capacity AutoMACS
25 /Box 2x108 Disposable 8,680원 LS 2x109 13,680원 XS 5 / Box 2x1010 135,400원 AutoMACS 5x2 /Box 4x109 Reusable 850원 ~1,700원

Major Advantages of AutoMACS
• 4x109 cells 을 5분안에 분리가능 • Low cell damage, high cell viability after separation • 9 preset programs ; target cells 특성에 따라 최적의 프로그램 선택 가능 • Input volume : 0.25mL~50mL • Reusable columns • Rare cells can be enriched to 10,000 fold • Whole blood 에서 직접 분리 가능 • Reproducible results • Standardization in sample processing

Features of AutoMACS Pro
Walk-away processing of up to six samples Automated sample uptake, fraction elution, system cleaning Direct whole blood isolation is possible up to 15 ml Sample cooling option Safe sensor-controlled process Optimized 10 programs for a wide variety of cell separations Based on renowned MACS Technology Compact benchtop design, also fits in laminar flow hood

MACS® Technologies - cell separation and beyond
Sample preparation Cell Separation Biomolecule Separation Cellular Analysis Cell Culture Molecular Analysis

MACS Products for Sample Preparation
Pre-Separation Filters Removal of cell clumps Dead Cell Removal Kit Depletion of dead cells Basic MicroBeads Removal of materials that bind unspecifically to MB Neural Tissue Dissoc. Kits Gentle and efficient enzymatic dissoc. of neural tissues PrepProtect Stab. Buffer RNA-stabilizer for fresh and frozen cells or tissues

- Miltenyi Biotec cares about customer needs -
GentleMACS - Miltenyi Biotec cares about customer needs - The quality of an experiment strictly depends on the quality of the sample preparation. In order to achieve optimum results. Miltenyi Biotec develops, manufactures and sells more than 800 products and services for biomedical research, particularly in the fields of cell biology, immunology, regenerative medicine and molecular biology. In addition to developing cellular therapies, Miltenyi Biotec is the leading provider of tools in this exciting area. Our contract manufacturing divisions manufacture custom biopharmaceuticals, cellular products and medical devices.

gentleMACS™ Programs and Protocols
Specific gentleMACS Protocols and Programs: C Tubes Dissociation of mouse spleen w/o collagenase treatment m_spleen_01 w/ collagenase treatment m_spleen_02, m_spleen_03 Dissociation of mouse brain including use of MACS® Neural Tissue Dissociation Kits m_brain_01, m_brain_02, m_brain_03 Dissociation of mouse liver with collagenase treatment m_liver_01, m_liver_02 M Tubes Isolation of total RNA from fresh or frozen tissue Isolation of mRNA from fresh or frozen tissue RNA_01, RNA_02

MACS Applications – Cell Separation
Immune Cell Study : T cells, B cells, NK cells, Monocytes, granulocytes, DCs Stem Cell Study : HSCs, MSCs for tissue regeneration, EPCs for angiogenesis, Cancer Stem Cells : CD133, CD326(EpCAM) ES Neuroscience

In-situ hybridization probes O4
gentleMACS CD133, Prominin-1 Indirect MicroBeads Neural Tissue Dissociation Kit (Papain) CD24 PSA-NCAM A2B5 NG2/MCSP MACSmolecular: PIQOR™ Microarrays SuperAmp™Service miRXplore™ Microarrays In-situ hybridization probes Neural Tissue Dissociation Kit (Trypsin) O4 Retinal Ganglion Cell Isolation Kit CD271 (LNGFR) MACSNeuro Medium (A) MACSNeuro Medium (E) Blue: existing products. Red: 2008 With our new products, our overall product portfolio is aiming at providing integrated solutions for NS Research With the addition of miRXploreTM microarray kit for Neuroscience (Q1, 2008) for microRNA analysis, and the in-situ hybridization probes we are building integrated solutions for research. Myelin Removal kit CD271 (LNGFR) CD11b

MACS Applications – Cell culture
Ready to use Media for human MSCs Quatification & Quality control • NH CFU-F Medium, 24x5 ml # MSC Expansion • NH Expansion Medium, 500 ml # MSC Differentiation • NH AdipoDiff Medium, 100 ml # • NH ChondroDiff Medium, 100 ml # • NH OsteoDiff Medium, 100 ml #

MACS Applications – Cell culture
HSC-CFU media: T cell Activation/Expansion Kit Cell expansion & differentiation bags Peptivator –CMV pp65

MACS Applications – Cell Analysis
antibodies for FACS analysis after MACS separation Kits for cell detection and enumeration Intracellular cytokine staining – CSA kits, FoxP-3 Immunofluorescence amplification – FASER kit Detection of epitope-tagged proteins – HA, c-myc, GFP

Cytokine Secretion Assay for Antigen Specific T cells Detection & Isolation

Antigen-specific T cells in disease
Infection Autoimmunity antigen-specific T cell Tumor Allergy Why are antigen-specific T cells of such a high interest? - They are key players in all specific immune responses. - They act as helper as well as effector cells in many diseases, like infections or malignancies. Analysis of the specific cells may provide important information on the cause of disease and development of treatment or vaccination. - Antigen-specific T cells can alsobe responsible for the development of some diseases, as autoimmunity, allergic reactions or alloreactivity. Here analysis of the responsible cells can help to cure or to prevent these disorders. - Analysis and isolation of antigen-specific T cells can yield important information on the specific cells, and can help to characterize the major antigens, responsible for the immune reactions. Alloreactivity MD

MACS Cytokine Secretion Assay - the principle
Catch Reagent secreted cytokine Anti-PE MicroBeads Detection Antibody cytokine secreting cell • • • • - Optionally the cytokine secreting cells can be enriched with Anti-PE MicroBeads. - For analysis of cytokine secreting cells with frequencies below %, sensitivity of the Cytokine Secretion Assay can be enhanced by magnetic enrichment of the cells. This enables for detection of cytokine secreting cells as low as 1 in a million. - The magnetic enrichment is also a useful tool for culturing and expansion of these cells, functional analyses or for other downstream- applications. antigen-specific restimulation labeling with Catch Reagent secretion period (optional) magnetic labeling MD

IFN-g secretion assay - procedure
To PBMC, add antigen for stimulation (3 - 16h, 37ºC) Analysis or culture • Add Catch Reagent • Secretion (45 min, 37ºC) • Add Detection Antibody • Magnetic labeling MACS Isolation Elution of IFN-g secreting cells (up to 10,000-fold enrichment) MD

and a variety of other applications
MACSmix for the secretion period and a variety of other applications - For continuous resuspension of the cells we developed the MACSmix. - The MACSmix runs on rechargeable batteries and works independently from permanent power supply. It can be placed in the incubator as well as in the refrgerator and is a helping hand in the lab for a variety of applications.

MACS Applications – Molecular study
From cell to gene expression profile and beyond µMACS Isolation mRNA/proteins mRNA stabilization Transcription factor isolation Identification of interaction partners Tag protein analysis Protein immunopurification µMACS mRNA isolation & in-column cDNA labeling µMACS mRNA isolation & in-column cDNA synthesis MultiMACS Microarray + Bioinformatics Services PIQOR™ DNA Microarrays, a-Hyb Station analysis e.g real-time PCR MACS Technology is well known and established for the isolation of cells. MACS Technology uses super-paramagnetic MicroBeads for magnetic labeling of cells and MACS Columns and Separators for separation. But this technology cannot only be used for pure and efficient cell isolation. It has been adjusted for molecular biology applications and provides similar advantages for the isolation of molecules as well as for their subsequent downstream applications, i.e. cDNA synthesis of mRNA molecules. Combined, you can concentrate on the cells you are interested in and specifically isolate the molecule you need for your downstream applications. Together with the PIQORTM Technologie, a-Hyb Station and the BioInformatics Service as well as the MultiMACS for High Throughput MACSmolecular products offer you the whole portfolio for gene expression profiling and beyond applications. In this presentation we will focus on the MACSmolecular products based on the MACS Technology. Cell transfection and MACSelect enrichment for functional gene expression Bioinformatics analysis

MACS Applications – Molecular study
a-HybTM Hybridizatin Station MultiMACS Separator

MACSelectTM System - The smart selection after each transfection
Many positives, low back ground Selection in 3 hours only No change of transfection method Choose the optimal surface marker for your cells Adherent and suspension cells applicable Stable and transient cells applicable

MACSelect surface markers
Free choice of best-fitting marker CD4 H-2Kk LNGFR • trypsin sensitive • CD4 is naturally expressed on T helper cells, monocytes and dendritic cells • requires co-expression of beta-2-microglobulin • H-2Kk expression is restricted to some rarely used mouse strains like AKR/J and CBA/Ca. • LNGFR is expressed in the CNS and PNS on autonomic and sensory neurons and glial cells, on bone marrow fibroblasts, follicular dendritic cells, some mesenchymal cells MD

Enriched transfected cells
Principle of MACSelect transfected cell selection Plasmid with DNA of interest pMACS Co- transfection Incubation Magnetic labeling Magnetic separation Enriched transfected cells MD

Notice! - 감사합니다 - MACS 모든 신제품에 한하여 Test Sample 제공
 학술 문의 : - 감사합니다 -

Magnetic Cell Separation

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