bd imag cell separation system · 2007. 3. 22. · recovery, the bd imag system has sufficient...

BD™ IMag Cell Separation System

2 www.bdbiosciences.com

Table of Contents

Magnetic Cell Separation with BD™ IMag Particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

BD IMag Enrichment Protocol Flow Chart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

BD IMag Enrichment Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Negative Selection versus Positive Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Sample Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

The Flexibility of the BD IMag Cell Separation System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Sample Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

BD IMag Sets for Enrichment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

BD IMag Reagents for Positive Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

For Research Use Only. Not for use in diagnostic or therapeutic procedures.All applications are either tested in-house or reported in the literature. See Technical Data Sheets for details.BD flow cytometers are class I (1) laser products.Purchase does not include or carry any right to resell or transfer this product either as a stand-alone product or as a component of another product. Any use of this product other than the permitted use without the express written authorization of Becton Dickinson and Company is strictly prohibited.BD, BD Logo and all other trademarks are the property of Becton, Dickinson and Company. ©2004 BD

Figure 1. BD IMagnet direct magnet system

The BD™ IMag Cell Separation System isbased on a simple yet highly effective directmagnet technology that allows for the rapidpositive and/or negative selection (enrichment)of cell populations without the use of magneticseparation columns. When using the BD IMagsystem, one can expect excellent purities andrecoveries of specific leukocyte subpopulations in a few short steps.

BD IMag particles are nanometer-sized super-paramagnetic particles with monoclonalantibody or streptavidin covalently bound to their surface. These particles have beenoptimized for both positive and negativeselection of leukocyte sub-populations usingthe BD™ IMagnet direct magnet (DM) system (figure 1).

Positive selections typically make use of BD IMag particles that have monoclonalantibodies covalently bound to their surface.These particles specifically target a leukocytesub-population of interest. Once placed withinthe BD IMagnet, targeted cells migrate towardthe magnet and are retained within the magneticfield while the unlabeled cells are drawn off.The targeted cells can then be collected andused in the desired application ater removalfrom the magnetic field. In the event thatnegative selection (enrichment) is required, the unlabeled cells are drawn off and can beutilized for a variety of applications such ascell sorting.

Magnetic Cell Separation with BD™ IMag Particles

4 www.bdbiosciences.comUnless otherwise specified, all products are for Research Use Only.

Not for use in diagnostic or therapeutic procedures. Not for resale.

BD™ IMag Enrichment Protocol Flow Chart

Enriched(depleted) fraction

Ready for analysis or culture

Final enriched fractionReady for Analysis or Culture

BD™ IMagnet:6-8 minutes

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Positive fractionReady for Analysis or Culture

BD™ IMag labeled cell suspension

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Figure 2. BD IMag Flexible Enrichment Protocol

1 Place the BD IMag labeled cell suspension onto the BD IMagnet.2- Remove the supernatant containing the negative fraction and place in an appropriately labeled tube.2+ Remove the tube containing the positive fraction.3 Resuspend the positive fraction and place back onto the BD IMagnet.4- Remove the negative fraction and pool with the negative fraction from step 2-.4+ Remove the tube containing the positive fraction.

5 Resuspend the positive fraction and place back onto the BD IMagnet.6- Remove the negative fraction and pool with the negative fraction from steps 2- and 4-.6+ Remove the positive fraction and resuspend the cells for use.7 Place the combined negative fractions on the BD IMagnet.8- Remove remaining twice enriched fraction and place in an appropriately labeled tube for use.8+ Remove and discard residual positive fraction.

Enrichment by depletion or negative selection is used for researchapplications which require a cell population with high levels ofpurity and no antibody or particles bound to their surface. In this instance, all unwanted cells are first labeled with a cocktailcontaining monoclonal antibodies against antigens expressed by all unwanted cells. After washing away unbound antibody, a second-step BD IMag reagent is used to magnetically label these cells. The labeled cells migrate to the BD IMagnet leavingbehind a pure and “untouched” sub-population of cells to be drawn off (figure 2).

BD Biosciences Pharmingen has significantly expanded the BD IMag product line. In addition to the human and mousepositive selection reagents, there are now human and mouseenrichment sets providing researchers with a cost-effective methodfor isolating untouched target cells with exceptionally high levelsof purity and recovery (figures 3 and 4). Finally, there are severalsecond-step BD IMag particle reagents that allow researchers tocustomize their magnetic cell separations using their own specificantibodies or antibody cocktails.

5Unless otherwise specified, all products are for Research Use Only.Not for use in diagnostic or therapeutic procedures. Not for resale.

* Comparison not available

Target Cell PurityBD IMag Mouse Enrichment Sets vs. Competitor

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BD™ IMag Enrichment Reagents

Figure 3a. A comparison of human target cell purity obtained after enrichment ofdifferent lymphocyte sub-populations from PBMC using either the BD IMag HumanEnrichment Sets or the competitor’s corresponding magnetic separation column products.

Figure 3b. A comparison of human target cell recovery obtained after enrichment ofdifferent lymphocyte sub-populations from PBMC using either the BD IMag HumanEnrichment Sets or the competitor’s corresponding magnetic separation column products.

Figure 4a. A comparison of mouse target cell purity obtained after enrichment ofdifferent lymphocyte sub-populations from spleen (or bone marrow for hematopoieticprogenitors) using either the BD IMag Mouse Enrichment Sets or the competitor’scorresponding magnetic separation column products.

Figure 4b. A comparison of mouse target cell recovery obtained after enrichment ofdifferent lymphocyte sub-populations from spleen (or bone marrow for hematopoieticprogenitors) using either the BD IMag Mouse Enrichment Sets or the competitor’scorresponding magnetic separation column products.

BD™ IMag Competitor

Negative Selection versusPositive SelectionPositive cell selections yield excellentresults with respect to purity, recovery,and viability of selected cells. However,depending on the cell type being selectedand the surface antigen being targeted bythe particle, positive selections can resultin cells becoming activated or otherwisefunctionally altered. Even though theprobability of activation is low, thismagnetic particle-induced activation maybe an issue for researchers whospecifically require purified yetunstimulated cells. As such, they shouldconsider negative selection for their cellseparations.

Negative selection is a simple enrichmentprocess by which unwanted cells aremagnetically labeled and removed leavingan untouched population of target cells.To demonstrate how little magneticparticle-induced activation occurs withnegative selection, mouse CD4 T cellswere isolated either by positive or negativeselection and then placed in culture for 48hours, at which time the surfaceexpression of activation markers CD25and CD69 were examined (figure 5).

CD4 T cells were either positively selectedfrom mouse spleen using BD IMag Anti-Mouse CD4 Particles – DM or enrichedusing the BD IMag Mouse CD4 TLymphocyte Enrichment Set – DM. Forflow cytometric analysis of the twoseparation techniques, unmanipulatedBALB/c splenocytes (figure 5A), thepositively selected CD4 T cells (figure 5B),and the enriched (negatively selected) CD4T cells (figure 5C) were stained with APCanti-CD3e (clone 145-2C11) and PE anti-CD4 (clone GK1.5). The percentCD3+/CD4+ cells in each sample is given.

Immediately after isolation, the purifiedCD4 T cells were either placed inuncoated wells containing normal culturemedia or they were suspended in wellspre-coated with anti-CD3 (clone 17A2)and containing media with soluble anti-CD28 (clone 37.51). After 48 hours inculture, the cells were stained with PEanti-CD25 (PC61) and FITC anti-CD69(H1.2F3) to detect activated lymphocytes,and propidium iodide to detect dead cells.Positively selected cells that had beensuspended in media alone had more deadcells when compared to the enrichedfraction (compare figures 5D and 5F) andof the viable cells, approximately 28%were CD25+ (figure 5E). In contrast, theenriched cells had approximately 5%CD25+ cells (figure 5G). In either case,very little CD69 expression was seen.When comparing the cells that had beencultured in the presence of anti-CD3 andanti-CD28, enriched cells still had agreater viability after 48 hours (comparefigures 5H and 5J), but there was virtuallyno difference in the expression pattern ofCD25 and CD69 (compare figures 5I and 5K).



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Figure 5. Expression of activation markers CD25 and CD69 after either positive selection or negative selection (enrichment) of CD4 T cells usingthe BD IMag Mouse CD4 Particles – DM and BD IMag Mouse CD4 T Lymphocyte Enrichment Set – DM respectively.

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While the basic enrichment (negativeselection) protocol (figure 2) has beenoptimized to maximize both purity andrecovery, the BD IMag system has sufficientflexibility to allow any separation to becustomized for a particular researcher’sneeds. For example, by reducing therecommended number of washes, evengreater purity can be obtained. Alternately,by increasing the number of washesand/or omitting the final incubation onthe BD IMagnet (figure 2, step 7) therecovery can be increased.

Another advantage of negative selectionsis the ability to do a positive selectionusing the enriched cells as a startingmaterial. This makes the isolation ofuncommon cell sub-populations possiblein just a few simple steps. The examplesbelow demonstrate how the simple BD IMag enrichment protocol can bemanipulated to increase purity and isolatespecific cell sub-populations within theCD4 T cell subset.

Maximizing the Purity of Enriched Cells

CD4 T cells were isolated from humanperipheral blood mononuclear cells(PBMC) using the BD IMag Human CD4T Lymphocyte Enrichment Set – DM. Forflow cytometric analysis, theunmanipulated PBMCs (figure 6A), the“combined once enriched” fraction (figure6B), and the “twice enriched” fraction(figure 6C), were stained with APC anti-CD4 (RPA-T4) to detect CD4 T cells anda mixture of PE anti-CD8 (RPA-T8),CD11b (ICRF44), CD16 (3G8), CD19(HIB19), CD36 (CB38), CD56 (B159),CD123 (7G3), glycophorin A (GA-R2),and γ/δ TCR (B1) to detect non-CD4 Tleukocytes and erythrocytes. The percentCD4 T cells in each sample is given.

The “once-enriched” CD4 T cell fractionwas obtained after 3 incubations on theBD IMagnet (steps 1 through 6-, figure 2).To increase purity, this enriched fractionwas placed on the BD IMagnet a finaltime to give a “twice-enriched” fraction(steps 7 through 8-, figure 2). In general,this additional incubation will result in a2%–20% increase in purity but mayreduce recovery by up to 10%. Factorsthat can influence purity and recoveryinclude the anti-coagulant used, thequality of the sample preparation, thedonor, the specific cell type beingenriched, and the percent target cells inthe unmanipulated sample.

In some instances, an even purer sampleof CD4 T cells is required. For example,when purifying T regulatory cells whichare CD4+/CD25+ it is preferred to haveno contaminating non-CD4 T cellspresent. In such cases we recommendeliminating all washes (no steps 3 and 5,figure 2) before obtaining the twice-enriched fraction. For the CD4 T cellenrichment, one can then expect puritiesgreater than 98%. However, recoveriesmay be up to 30% lower. In the exampleshown here, CD4 T cells were firstenriched from PBMC using the BD IMagHuman CD4 T Lymphocyte EnrichmentSet – DM , followed by a CD25 positiveselection using the BD IMag Anti-HumanCD25 particles - DM. UnmanipulatedPBMC (figure 6D), the CD4 enrichedfraction (figure 6E, prepared with nowash steps), and the T regulatoryCD4+/CD25+ enriched fraction (figure6F), were stained with PE anti-CD4 (RPA-T4) and APC anti-CD25 (M-A251). Thepercent total CD4+ cells and the percentCD4+/CD25+ T regulatory cells in eachsample are given.

Subpopulation Selection from Enriched Cells

CD4 T cells can also be divided into naïvecells that have not seen antigen andexpress CD45RA and those cells that haveseen antigen (activated or memory) andexpress CD45RO. Both of these CD4 Tcell subsets can be isolated to high levelsof purity by first doing a CD4 enrichmentfollowed by a CD45RO positive selection.Since CD45RO and CD45RA are almostmutually exclusive on CD4 T cells, theCD45RO-negative fraction would beexpected to contain the CD45RA+expressing cells.

Again, CD4 T cells were first enrichedfrom PBMC using the BD IMag HumanCD4 T Lymphocyte Enrichment Set –DM, followed by a CD45RO positiveselection using the BD IMag Anti-HumanCD45RO Particles – DM. For flowcytometric analysis, enriched CD4 T cells(figure 6G), the CD45RO positivefraction (figure 6H) and the CD45ROnegative fraction (figure 6I) were stainedwith APC anit-CD4 (RPA-T4) and FITCanti-CD45RO (UCHL1). The percentCD4+/CD45RO+ cells in each sample are given.

To show that the CD45RO negativefraction contained the CD45RA+ cells,this fraction was also stained with APCanti-CD4 (RPA-T4) and FITC anti-CD45RA (HI100) (figure 6J). The percentCD4+/CD45RA+ cells is given. Therefore,by combining the use of the CD4 T cellenrichment set with the CD45RO positiveselection reagent, it was possible to isolatehighly purified populations ofCD4+/CD45RO+ and CD4+/CD45RA+cells in just a few simple steps.

The Flexibility of the BD™ IMag Cell Separation System


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Figure 6. Demonstration of how the basic enrichment protocol can be manipulated for different experimental needs and how positive selectionscan be coupled with enrichments to isolate uncommon cell subpopulations.



B Lymphocyte Enrichment Set - DM Hu Biotinylated antibody cocktail and BD IMag Streptavidin Particles Sep BD IMag-DM 1 x 109 Cells May 2004

CD4 T Lymphocyte Enrichment Set - DM Hu Biotinylated antibody cocktail and BD IMag Streptavidin Particles Sep BD IMag-DM 1 x 109 Cells 557939

CD8 T Lymphocyte Enrichment Set - DM Hu Biotinylated antibody cocktail and BD IMag Streptavidin Particles Sep BD IMag-DM 1 x 109 Cells 557941

NK Cell Enrichment Set - DM Hu Biotinylated antibody cocktail and BD IMag Streptavidin Particles Sep BD IMag-DM 1 x 109 Cells May 2004

T Lymphocyte Enrichment Set - DM Hu Biotinylated antibody cocktail and BD IMag Streptavidin Particles Sep BD IMag-DM 1 x 109 Cells 557874

BD™ IMag Sets for EnrichmentHuman

DESCRIPTION REACT CONTENTS APPS FORMAT SIZE CAT. NO.

B Lymphocyte Enrichment Set - DM Ms Biotinylated antibody cocktail and BD IMag Streptavidin Particles Sep BD IMag-DM 1 x 109 Cells 557792

CD4 T Lymphocyte Enrichment Set - DM Ms Biotinylated antibody cocktail and BD IMag Streptavidin Particles Sep BD IMag-DM 1 x 109 Cells 558131

CD8 T Lymphocyte Enrichment Set - DM Ms Biotinylated antibody cocktail and BD IMag Streptavidin Particles Sep BD IMag-DM 1 x 109 Cells 558471

Dendritic Cell Enrichment Set - DM Ms Biotinylated antibody cocktail and BD IMag Streptavidin Particles Sep BD IMag-DM 1 x 109 Cells 557955

Hematopoietic Progenitor Cell Ms Biotinylated antibody cocktail and BD IMag Streptavidin Particles Sep BD IMag-DM 1 x 109 Cells 558451Enrichment Set - DM

NK Cell Enrichment Set - DM Ms Biotinylated antibody cocktail and BD IMag Streptavidin Particles Sep BD IMag-DM 1 x 109 Cells 557954

T Lymphocyte Enrichment Set - DM Ms Biotinylated antibody cocktail and BD IMag Streptavidin Particles Sep BD IMag-DM 1 x 109 Cells 557793

Mouse

DESCRIPTION REACT CONTENTS APPS FORMAT SIZE CAT. NO.

BD™ IMag Sets for Enrichment

DM - for use with the BD IMagnet Direct MagnetMSC - for use with Magnetic Separation ColumnsBD IMag particles are prepared from Carboxy-functionalized magnetic particles manufactured by Skold Technology


DM - for use with the BD IMagnet Direct MagnetMSC - for use with Magnetic Separation ColumnsBD IMag particles are prepared from Carboxy-functionalized magnetic particles manufactured by Skold Technology

CD3 Magnetic Particles - DM Hu HIT3a Sep BD IMag-DM 1 x 109 Cells 552593

CD3 Magnetic Particles - MSC Hu HIT3a Sep BD IMag-MSC 1 x 109 Cells 552594

CD4 Magnetic Particles - DM Hu, Rhe L200 Sep BD IMag-DM 1 x 109 Cells 557767

CD4 Magnetic Particles - MSC Hu, Rhe L200 Sep BD IMag-MSC 1 x 109 Cells 557768

CD8 Magnetic Particles - DM Hu, Rhe SK1 Sep BD IMag-DM 1 x 109 Cells 557766

CD8 Magnetic Particles - MSC Hu, Rhe SK1 Sep BD IMag-MSC 1 x 109 Cells 557765

CD14 Magnetic Particles - DM Hu, Rhe MφP9 Sep BD IMag-DM 1 x 109 Cells 557769

CD14 Magnetic Particles - MSC Hu, Rhe MφP9 Sep BD IMag-MSC 1 x 109 Cells 557770

CD19 Magnetic Particles - DM Hu HIB19 Sep BD IMag-DM 1 x 109 Cells 551520

CD19 Magnetic Particles - MSC Hu HIB19 Sep BD IMag-MSC 1 x 109 Cells 551521

CD25 Magnetic Particles - DM Hu 2A3 Sep BD IMag-DM 1 x 109 Cells 558005

CD45RA Magnetic Particles - DM Hu HI100 Sep BD IMag-DM 1 x 109 Cells 557981

CD45RO Magnetic Particles - DM Hu UCHL1 Sep BD IMag-DM 1 x 109 Cells 557986

CD56 Magnetic Particles - DM Hu, Rhe NCAM16.2 Sep BD IMag-DM 1 x 109 Cells 557775

γ/δT Cell Receptor Separation Set - DM Hu B1 Sep BD IMag-DM 1 x 109 Cells May 2004

Human

DESCRIPTION REACT CLONE APPS FORMAT SIZE CAT. NO.

CD4 Magnetic Particles - DM Ms GK1.5 Sep BD IMag-DM 2 x 109 cells 551539

CD4 Magnetic Particles - MSC Ms GK1.5 Sep BD IMag-MSC 2 x 109 cells 551540

CD8a Magnetic Particles - DM Ms 53-6.7 Sep BD IMag-DM 2 x 109 cells 551516

CD11b Magnetic Particles - DM Ms M1/70 Sep BD IMag-DM 2 x 109 cells 558013

CD11b Magnetic Particles - MSC Ms M1/70 Sep BD IMag-MSC 2 x 109 cells 558011

CD45R/B220 Magnetic Particles - DM Ms RA3-6B2 Sep BD IMag-DM 2 x 109 cells 551513

CD90.2 (Thy1.2) Magnetic Particles - DM Ms 30-H12 Sep BD IMag-DM 2 x 109 cells 551518

Ly-6G and Ly-6C (Gr-1) Magnetic Particles - DM Ms RB6-8C5 Sep BD IMag-DM 2 x 109 cells 558111

Ly-6G and Ly-6C (Gr-1) Magnetic Particles - MSC Ms RB6-8C5 Sep BD IMag-MSC 2 x 109 cells 558012

NK Cell Separation Set - DM Ms DX5 Sep BD IMag-DM 2 x 109 cells May 2004

Mouse


Allophycocyanin (APC) Magnetic Particles - DM E30-221 Sep BD IMag-DM 1 x 109 cells 557932

R-Phycoerythrin (PE) Magnetic Particles - DM E31-1459 Sep BD IMag-DM 1 x 109 cells 557899

Mouse IgG1 Magnetic Particles - DM A85-1 Sep BD IMag-DM 1 x 109 cells 557983

Rat Ig, κ light chain Magnetic Particles - DM MRK-1 Sep BD IMag-DM 1 x 109 cells 558002

Streptavidin Magnetic Particles Plus - DM Sep BD IMag-DM 1 x 109 cells 557812

Streptavidin Magnetic Particles Plus - MSC Sep BD IMag-MSC 1 x 109 cells 557811

Buffer (10x) for use with BD™ IMag cell separation products Sep Buffer 100 ml 552362

BD™ IMagnet Cell Separation Magnet Sep Magnet 1 each 552311

Other


BD™ IMag Reagents for Positive Selection

Visit www.bdbiosciences.com/bdimag for additional product details.

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bd imag cell separation system · 2007. 3. 22. · recovery, the bd imag system has sufficient...

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