Population Enrichment and Isolation with Magnetic Sorting

by Joshua M. Kogot, Joseph M. Pennington, Deborah A. Sarkes, 

Dimitra N. Stratis-Cullum, and Paul M. Pellegrino

ARL-TN-0452 September 2011

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Army Research Laboratory

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ARL-TN-0452 September 2011

Population Enrichment and Isolation with Magnetic Sorting

Joshua M. Kogot, Joseph M. Pennington, Deborah A. Sarkes, 

Dimitra N. Stratis-Cullum, and Paul M. Pellegrino

Sensors and Electron Devices Directorate, ARL

Approved for public release; distribution unlimited. 
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Population Enrichment and Isolation with Magnetic Sorting

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Joshua M. Kogot, Joseph M. Pennington, Deborah A. Sarkes, 

Dimitra N. Stratis-Cullum, and Paul M. Pellegrino

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14. ABSTRACT

Manual and semi-automated sorting methods (MACS) provide a high-throughput, less costly method for sample enrichment 

compared to fluorescence activated cell sorting (FACS). Magnetic sorting methods use commercially available magnetic beads 

with many surface modifications available for customizable sorting methods. In this note, we outline the methods for magnetic 

cell sorting using manual and benchtop magnets (MACS), as well as a micromagnetic sorter (MMS) that uses diposable, 

microfluidic cartridges. Along with magnetic sorting methods, we detail flow cytometry analysis techniques to quantify cell 

population enrichment for isolation of bacterial display library reagents. 

15. SUBJECT TERMS

MACS, magnetic sorting, display library

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Joshua M. Kogot

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iii

Contents

List of Figures iv

List of Tables iv

Foreword v

Introduction 1

Materials and Methods 1

Results and Discussion 7

Conclusion 9

References 10

List of Symbols, Abbreviations, and Acronyms 12
iv

List of Figures

Figure 1. Typical enrinchment results for magnetic sorting using MMS. The increase in the 

PE signal (green) in each round (1-3, left to right) indicates an enriched positive 

population. The positive increase is noted on the x-axis (PE-H) in the histograms in the 

top panel and along the y-axis in the dot plot in the bottom panel. The red signal in each 

plot is the background cell fluorescence measured in the PE emission channel. Either a 

histogram of PE-H vs. count or a dot-plot with FITC-A vs. PE-A is typical to show 

population enrichment by flow cytometry.................................................................................8

List of Tables

Table 1. Typical parameters for magnetic sorting with MMS or manual MACS using 

bacterial display libraries as detailed in the text. .......................................................................4
v

Foreword

The purpose of this report is to outline procedures and methods as a guide for magnetic sorting. 

The protocol presented has been established from a previously funded ICB 6.2 research project 

funded through the Army university-affiliated research center (UARC), the Institute for 

Collaborative Biotechnologies (University of California, Santa Barbara under grant DAAD19-

03-D-0004), current mission work in synthetic recognition elements, and a current 6.1 research 

project funded by the Defense Threat Reduction Agency (DTRA, Grant no. BRCALL08-Per3-P2-0028) for smart affinity reagent development. These methods can be used directly for 

magnetic sorting or can serve as a guideline for developing new magnetic sorting methods.
vi

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1

Introduction

Magnetic sorting is a convenient, low-cost method used to isolate antibody alternatives 

(magnetic activated cell sorting [MACS]) and is often used to enrich cell populations prior to 

fluorescence activated cell sorting (FACS), due to the high-throughput achieved with MACS (1). 

There are a number of current magnetic sorting methods. Manual MACS uses a benchtop, rareearth magnetic that is typically housed in plastic and can be purchased for different tube sizes. 

The autoMACS (Miltenyi Biotech) is semi-automated and is marketed for sorting against 

eukaryotic cells, including yeast. The autoMACS uses a disposable magnetic column for 

trapping cells labeled with magnetic beads. Recently, the U.S. Army Research Laboratory 

(ARL) transitioned a microfluidic magnetic sorter (MMS) from Cynvenio Biosystems during an 

ICB 6.2 project that was developed as a low-cost magnetic sorter for sorting against bacteria 

display libraries (2). The MMS uses disposable microfluidic cartridges, relying on a highsurface area trapping region for efficient cell capture. Besides being used for bacterial library 

sorting, the MMS can also be used for eukaryotic cell sorting and protein purification using 

magnetic beads. 

Herein, we describe the procedures for employing magnetic sorting for bacterial display library 

enrichment, using either MMS or manual MACS. The following method was developed using a 

bacterial display library expressing a random 15-amino acid peptide on the outer membrane 

surface (3–5). This method has also been used, with minor modifications, to sort yeast display 

libraries: yeast are larger cells, which results in greater sample volumes for an equal number of 

cells, and sorting with yeast requires longer growth periods because of the slower doubling rate. 

For a review on autoMACS sorting with yeast libraries see Chao, et al. 2006 (6). This outlined 

procedure will be useful for researchers new to library sorting procedures using manual MACS 

or a semi-automated sorter, such as the MMS. 

Materials and Methods

Bacterial Culture Growth Materials: 

Luria Broth (LB) (Fisher Scientific) supplemented with 25 g/ml Chloramphenicol (Sigma) is 

used for all bacteria growth cultures, typically 15 g/L, and autoclaved prior to use (LB-Cm25). 

For surface display induction, a 4% w/v L-arabinose stock (2.0 g in 50 mL Sigma) is used to 

supplement growth media, while a 20% w/v D-glucose solution (10.0 g in 50 mL H2O) is used to 

inhibit induction during overnight growth and recovery steps. Both the arabinose and glucose 

solutions are sterile-filtered prior to use. LB-Cm25 agar are made, in house, using LB and agar 
2

combined in solution, autoclaved, and cooled to approximately 45 °C before the addition of 

chloramphenicol to final concentration of 25 g/mL.

MMS and MACS Sorting Materials:

A PBS solution, phosphate buffered saline, pH 7.4, is used as the binding buffer and PBS 

containing Bovine serum albumin (BSA) at 0.5% w/v final concentration is used as the wash 

buffers. For this procedure, the magnetic beads being used are Dynabeads® MyOne Strepavidin 

T1 beads (Invitrogen). The T1 beads are recommended for protein, biopanning, and cell 

isolation, and are compatible with BSA. Other Dynabead variants include M-280, M-270, and 

C1, and each bead ranges in size and suggested application (Invitrogen). Magnetic beads of 

varying sizes or surface modifications are available from other suppliers (Thermo Fisher 

Scientific, Miltenyi, Biorad, and more). For target coupling to T1 streptavidin beads, a 

biotinylation step is necessary. Biotin labeling kits are available from many suppliers (Pierce, 

Invitrogen, Qiagen, etc.) for labeling proteins with biotin through free amine moieties on surface 

exposed lysine residues. Unreacted biotin can be removed by desalting columns, dialysis, or gel 

filtration, or other method recommended by the biotinylation kit manufacturer.

Flow Cytometer Analysis Materials:

Streptavidin-R-Phycoerythrin (SAPE) and Neutravidin-R-Phycoerythrin (NAPE) from 

Invitrogen are used to fluorescently label biotinylated targets already bound to the cell surface. 

SAPE and NAPE are typically used at a 1:200 dilution of SAPE/NAPE:PBSB, and have 

excitation wavelength of 480, 545, and 565 nm, and an emission wavelength of 578 nm. 

Magnetic Sorting Methods:

Initial Sort (Round 1):

1. Obtain bacterial library (eCPX or other similar library) and amplify library by growing 

culture in LB Cm25 overnight and aliquoting at least 3x1011 cells/tube, and storing at 

–80 °C.

2. Add one frozen aliquot into 500 mL of LB-Cm25 (LB + 25μg/mL Chloramphenicol) and 

grow at 37 °C with shaking to an OD600 (optical density at 600 nm) of 0.55–0.70. This 

growth step typically requires 1 hr and 45 min. 

3. Once grown to the correct OD600, induce the library with final concentration of 0.04% 

Arabinose (5 mL of 4% Arabinose stock solution in 500 mL) and incubate for 35 min at 

37 °C. Check OD600 after induction. At 1 OD600, there are approximately1x109cells/mL.

4. Centrifuge 2x1011 cells (table 1, cells for sorting Round 1) at 5000 x g for 20 min.

5. Remove supernatant and resuspend cells in 1.5 mL of PBS buffer. To resuspend pellet, 

gently swirl until dissolved; DO NOT VORTEX.
3

Library Depletion with Magnetic Beads (Negative Sort)

1. Resuspend cell pellet in 1 mL of 109 washed Strepavidin T1 beads.

a. 109beads is 100 μL of stock solution beads, wash by resuspending beads in 1 mL of 

PBS then centrifuging at 5000 x g for 5 min, and finally resuspending in 1 mL of PBS. 

2. Incubate cell suspension for 15 min at room temperature with gentle rotation.

3. Depletion by Magnetic Sorting:

a. MMS instrument...Run the MMS “1st negative selection” (Day 1-1st negative 

Selection on software) to remove any cells that bind to the beads alone.

b. Manual MACS

i. Using a microcentrifuge magnet separator, place the 1.5 mL microcentrifuge tube 

against the magnet. 

ii. After 5 min, remove media that contains unbound cells and wash beads with 1 mL of 

PBSB by inverting the tube 3–4 times in the absence of the magnet before returning 

the sample to the magnet for subsequent wash steps. Combine all wash samples 

with original unbound cell sample as streptavidin depleted library. After three

washes, discard the magnetic beads.

4. Centrifuge the positive collection tube (containing the depleted library) at 5000 x g for 

5 min, then remove supernatant and resuspend in 1 mL of PBS.

Positive Sorting Round 1 after Depletion

1. Transfer cells to a 2 mL centrifuge tube and incubate with biotinylated target at 4 °C with 

rotation for 45 min. The starting concentration for each target is typically 600 nM in PBS 

(table 1) but may be increased to a ~1 μM starting concentration if low enrichment is not 

seen after 2–3 rounds.
4

Table 1. Typical parameters for magnetic sorting with MMS or manual MACS using bacterial display 

libraries as detailed in the text.

2. Centrifuge cells at 5000 x g for 5 min then remove supernatant.

3. Resuspend cell pellet in 1 mL of 109 washed Streptavidin T1 beads. Approximate volume 

of sample is 2 mL with bead solution and cell pellet.

4. Incubate the cells at 4 °C for 30 min with rotation to allow binding between the beads and 

biotinylated target. 

5. Magnetic Sorting (MMS or manual MACS) 

a. MMS

i. Prepare PBSB wash buffers and mixing buffers (table 1). 

ii. Run the MMS “1st Round positive selection” (Day 1-1

st Positive Selection on 

software) to sort the library.

iii. When inserting the syringes onto the MMS card, ensure that a firm connection has 

been made without pressing down too hard. If leaking of the buffer solutions is 

seen above the MMS card after the sort has finished, the syringes may not have 

been secured well enough. If leaking occurs below the MMS card, back pressure 

Round 1 Round 2 Round 3 Round 4 

Cells for Sorting 10-times the 

library 

diversity 

(2 x 1011 for 

eCPX library)

5 - 10-times the 

depleted library 

diversity 

(at least 1x108cells 

for bacterial display)

5 - 10-times the 

depleted library 

diversity 

(at least 1x108cells 

for bacterial display)

5 - 10-times the 

depleted library 

diversity 

(at least 1x108cells 

for bacterial display)

T1 Magnetic 

Beads 100 l 15 l 8 l

4 l

Typical Target 

Concentration 600 nM 300 nM 150 nM 75 nM

Sample Volume

Mixing Buffer 

Vol.

Wash Buffer Vol.

2 mL,

3 mL,

8 mL

500 L

800 L

8 mL

500 L

800 L

8 mL

500 L

800 L

8 mL

Typical Plating 

Dilutions

1:10,000 

1:100,000 

1:1,000,000

1:100 

1:1000 

1:10,000

1:10 

1:100 

1:1000

1:2 

1:10 

1:100
5

from the syringes being too tight may have caused a rupture in the fluidics 

membrane.

iv. After the sort has been completed, place both the positive and negative collection 

tubes against a magnet to ensure all of the magnetic beads have been retained in the 

positive collection tube. 

1. If a significant amount of beads has not been recovered in the positive or 

negative sort, they may have been trapped on the MMS card, in which case the 

sort may need to be repeated with a new card. 

2. Continue with post-sorting processing with positive sample.

b. Manual MACS

i. Using a multicentrifuge magnet separator, place the 1.5 mL microcentrifuge tube 

against the magnet. 

ii. Sample washing (repeat 3 times). After 5 min, remove the solution and wash the 

beads with 1 mL of PBSB by inverting the tube 3–4 times in the absence of the magnet 

before returning the sample to the magnet for subsequent wash steps. After three

washes, resuspend the beads with 1 mL PBSB for post-sorting processing. 

Post-sorting Process for all Sorting Rounds

1. Warm 4 LB-Agar Cm25 plates for 2 h at 37 °C.

2. Remove a 10 L sample from the positive collection tube and dilute in PBS. (See table for 

plating dilutions).

3. Plate 100 L of each sample dilution and a 100 L sample of buffer alone on LB-Cm25-

Agar plates.

4. Add the remaining positive sample to a 5 mL LB-Cm25 culture containing 0.2% glucose, 

final concentration, using a 20% stock glucose solution. 

Sorting after Overnight Growth (Round 2–4 sorting)

1. Passage a fresh bacteria culture from overnight growth culture using 100 L in 5 mL LBCm25 without glucose. *Glucose inhibits the induction with arabinose. 

2. Grow cells at 37 °C for approximately 1 h and 45 min; begin checking the OD600 after 1 h.

3. Bacteria culture with 500 L of 30% v/v Glycerol:H2O stock (final glycerol concentration 

of 15%).
6

4. Once grown to OD600 of 0.55–0.7, induce the library with a final concentration of 0.04% 

arabinose and incubate for 35 min at 37 °C. Check OD600 at induction completion. At 1 

OD600, there are approximately1x109cells/mL.

5. During induction, prepare flow cytometer labeling solutions (See Flow Cytometer Labeling 

section).

6. After induction, centrifuge cells for sorting (table 1, cells for sorting Round 2–4) at 5000 x 

g for 5 min. 

7. Optional. Negative sorting with Streptavidin T1 beads in round 2. 

a. Wash 1x108 Streptavidin T1 beads in 1 mL PBSB, centrifuge beads at 5000 x g for 

5 min, and resuspend in 50 L PBS. 

b. Run MMS (Day 2, 1st Negative Selection) using 1.5 mL microcentrifuge tube to collect 

cells, instead of 15 mL Falcon tube. 

8. Remove supernatant and resuspend cells in 50 L of PBS buffer containing biotinylated 

target at the desired concentration (table 1, typical target concentration Round 2–4). To 

resuspend pellet, gently swirl until dissolved; do not vortex.

9. Incubate the cells with biotinylated target protein on ice for 45 min.

10. Wash magnetic beads (table 1, magnetic beads for Round 2–4) with 1 mL PBS, centrifuge 

at 5000 x g for 5 min, and resuspend beads in 50 L PBS1. (100 L of Streptavidin T1 

beads is approximately 109 beads.)

11. Centrifuge sample at 5000 x g for 5 min, decant supernatant, and resuspend pellet in 50 L 

solution of magnetic beads.

12. Incubate sample and magnetic beads on ice for 30 min.

13. Adjust sample volume to 500 L with PBSB for sorting (See table 1, Sample Vol.).

14. Magnetic Sorting

a. Prepare mixing buffer volume and wash buffer volume using PBSB, according to table 

1.

b. Run the MMS positive selection using Day 2 for round 2 sorting and Day 3 for round 3 

and 4 sorting. Continue with post-sorting processing. (See Post Sorting Process 

Section). 

c. Manual MACS

i. Using a multicentrifuge magnet separator, place the 1.5 mL microcentrifuge tube 

against the magnet. In rounds 2–4, 0.5 mL of PBSB is used for washing. 
7

ii. In subsequent sorting rounds, it may be difficult to visualize the magnetic beads with 

the magnetic microcentrifuge tube holder. If this occurs, the wash steps should be 

performed by centrifugation (5000 x g for 5 min) instead of using manual MACS. 

v. After three washes, resuspend the beads with 1 mL PBS for post-sorting processing. 

15. Perform post-sorting processing.

Flow Cytometer Labeling

1. After induction, remove 5 L of induced cells, and incubate with a 25 L solutions of PBS 

containing 250 nM solution of biotinylated target, SAPE (positive control), NAPE (positive 

control), and 25 L of PBS alone for negative control. Incubate the samples on ice for 45 

min. 

2. After 45 min of incubation, centrifuge the samples at 5000 x g for 5 min, decant the 

supernatant, and begin measuring the SAPE, NAPE, and cells alone (negative control) on 

the flow cytometer. For flow cytometry analysis, each pellet should be resuspended in 0.5–

1.0 mL of BD FacsFlow®. The biotinylated target should be resuspended in a 25 L, 

250 nM solution of SAPE/NAPE for 30 min on ice for fluorescent labeling. 

3. After the 30-min incubation, centrifuge the sample at 5000 x g for 30 min and measure the 

percent fluorescence of the cells labeled with the biotinylated target. This value indicates 

the population enrinchment in each round. 

Results and Discussion

A typical magnetic sort shows positive population enrinchment with each round. In figure 1, the 

population enrichment results are shown using flow cytometry to evaluate the library after each 

sort. The percent positive population is displayed in green on each plot in figure 1, while the red 

populations are cells without PE on the surface. Most flow cytometers have excitation and 

emission filters, and optimized settings for PE fluorescence; therefore, PE is a common choice 

for flow cytometry. Alternatively, amine reactive fluorescent dyes or quantum dots can be 

directly coupled to the target for direct detection by flow cytometry to avoid non-specific binding 

of neutravidin or streptavidin (7, 8), or PE, which is a large, 240-kDa fluorescent protein from 

marine algae (9, 10). 
8

Figure 1. Typical enrinchment results for magnetic sorting using MMS. The increase in the PE signal (green) in each 

round (1-3, left to right) indicates an enriched positive population. The positive increase is noted on the x-axis 

(PE-H) in the histograms in the top panel and along the y-axis in the dot plot in the bottom panel. The red 

signal in each plot is the background cell fluorescence measured in the PE emission channel. Either a 

histogram of PE-H vs. count or a dot-plot with FITC-A vs. PE-A is typical to show population enrichment by 

flow cytometry. 

After one round of sorting, the increase in the PE signal indicates that 9.6% of the cells measured 

are positive for the sorted target. The number of positive cells increases from 9.6% to 23.4% to 

53.8% after round 3. The total enriched population increase per round of sorting is a strong 

indication that the sorting was successful, and a high affinity binder is likely. The sorting is 

concluded when the library enrichment does not change between each round. For bacterial 

display, this can occur after 3 rounds of sorting, while most sorts with the MMS do require 4 

rounds. 

The greatest advantage of magnetic sorting is that populations of 109– 1010 cells or greater can 

be sorted, compared to an upper limit of 108 cells per sort using FACS (11). As noted in table 1, 

it is advisable to initiate cell sorting with a 10-times oversampling of the library; therefore, 

FACS would be limited to libraries that have a diversity of 107or less. This limitation of FACS 

is problematic for sorting libraries that can have diversities of 1010 or greater such as bacterial, 

mRNA, and ribosomal display (11, 12). Using a microfluidic-based sorter such as MMS extends 

the sorting throughput to beyond 1010 cells per sort, which is typically the limit of magnetic 

sorting. 
9

Conclusion

Magnetic sorting using MMS or manual MACS is currently the most widely used method for 

library depletion for libraries with diversities greater than 107unique members. Magnetic sorting 

is a low-cost alternative to FACS, since library depletion can be accomplished using a strong 

magnet on the benchtop. The procedures and methods presented in this report can serve as a 

guide to design experiments for library depletion, including methods to evaluate library 

enrichment using flow cytometry. 
10

References

1. Siegel, R. W.; Coleman, J. R.; Miller, K. D.; Feldhaus, M. J. High Efficiency Recovery and 

Epitope-specific Sorting of an scFv Yeast Display Library. J Immunol Methods 2004, 286

(1-2), 141–53.

2. Stratis-Cullum, D. N.; Kogot, J. M.; Pellegrino, P. M. Rapid Peptide Reagent Isolation in a 

Disposable Microfluidic Cartridge; Army Research Laboratory: 2010; p 19.

3. Daugherty, P. S. Protein Engineering with Bacterial Display. Curr. Opin. Struct. Biol. 2007,

17, 474–480.

4. Rice, J. J.; Schohn, A.; Bessette, P. H.; Boulware, K. T.; Daugherty, P. S. Bacterial Display 

Using Circularly Permuted Outer Membrane Protein OmpX Yields High Affinity Peptide 

Ligands. Protein Sci 2006, 15 (4), 825–36.

5. Bessette, P. H.; Rice, J. J.; Daugherty, P. S. Rapid Isolation of High-affinity Protein Binding 

Peptides Using Bacterial Display. Protein Eng Des Sel 2004, 17 (10), 731–9.

6. Chao, G.; Lau, W. L.; Hackel, B. J.; Sazinsky, S. L.; Lippow, S. M.; Wittrup, K. D. Isolating 

and Engineering Human Antibodies Using Yeast Surface Display. Nat Protoc 2006, 1 (2), 

755–68.

7. Alon, R.; Bayer, E. A.; Wilchek, M. Cell-adhesive Properties of Atreptavidin are Mediated 

by the Exposure of an RGD-like RYD Site. Eur J Cell Biol 1992, 58 (2), 271–9.

8. Alon, R.; Bayer, E. A.; Wilchek, M. Streptavidin Contains an RYD Sequence Which 

Mimics the RGD Receptor Domain of Fibronectin. Biochem Biophys Res Commun 1990, 

170 (3), 1236–41.

9. Parks, D. R.; Herzenberg, L. A. Fluorescence-activated Cell Sorting: Theory, Experimental 

Optimization, and Applications in Lymphoid Cell Biology. Methods Enzymol 1984, 108, 

197–241.

10. Kronick, M. N.; Grossman, P. D., Immunoassay Techniques with Fluorescent 

Phycobiliprotein Conjugates. Clin Chem 1983, 29 (9), 1582–6.
11

11. Park, S. J.; Cochran, J. R. Protein Engineering and Design; CRC Press: Boca Raton, 2010; 

p 23–50.

12. Lipovsek, D.; Pluckthun, A. In-vitro Protein Evolution by Ribosome Display and mRNA 

Display. J Immunol Methods 2004, 290 (1-2), 51–67.
12

List of Symbols, Abbreviations, and Acronyms

ARL U.S. Army Research Laboratory

BSA bovine serum albumin

Cm25 Chloramphenicol at 25 g/mL

LB Luria Broth

FACS fluorescence activated cell sorting

MACS magnetic activated cell sorting

MMS microfluidic magnetic sorter

PBSB phosphate buffered saline with 0.5% BSA

SAPE Streptavidin-R-phycoerythrin

NAPE Neutravidin-R-phycoerythrin
13

NO. OF

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1 ADMNSTR

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THE PENTAGON

WASHINGTON DC 20301-3080

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CMND

ARMAMENT RSRCH DEV & ENGRG 

CTR 

ARMAMENT ENGRG & TECHNLGY 

CTR

ATTN AMSRD AAR AEF T 

J MATTS

BLDG 305

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3 CDS ATTN IMNE ALC HRR MAIL & 

RECORDS MGMT

ATTN RDRL CIO LL TECHL LIB 

ATTN RDRL CIO MT TECHL PUB

ATTN RDRL SEE G WOOD

ATTN RDRL SEE O 

D STRATIS-CULLUM (10 HCS, 1 CD)

ATTN RDRL SEE O J KOGOT 

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ATTN RDRL SEE O N FELL

ATTN RDRL SEE O P PELLEGRINO 

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14

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