Human granulocyte and monocyte isolation procedures...

Human granulocyte and monocyte isolation procedures: impact on 1

functional studies 2

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Lu Zhou1,2#, Rajesh Somasundaram3#, Rosa F. Nederhof1, Gerard Dijkstra1, Klaas Nico 4

Faber1, Maikel P. Peppelenbosch3, Gwenny M. Fuhler3* 5

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1Department of Gastroenterology and Hepatology, University Medical Center Groningen, 7

University of Groningen, Groningen, the Netherlands 8

2Department of Gastroenterology and Hepatology, Tianjin Medical University General 9

Hospital, Tianjin Medical University, Tianjin, P.R. China 10

3Department of Gastroenterology and Hepatology, Erasmus Medical Center, Erasmus 11

University of Rotterdam, Rotterdam, the Netherlands 12

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# These authors contributed equally 14

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* Correspondence to: Dr. Gwenny M. Fuhler, Department of Gastroenterology and 16

Hepatology, Erasmus Medical Center, Erasmus University of Rotterdam, ‘s 17

Gravendijkwal 230, 3015CE, Rotterdam, the Netherlands; Email: [email protected]; 18

Tel: +31-10-7035821; Fax: +31-10-7031793 19

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Running title: Comparison of granulocyte and monocyte isolation methods 21

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Copyright © 2012, American Society for Microbiology. All Rights Reserved.Clin. Vaccine Immunol. doi:10.1128/CVI.05715-11 CVI Accepts, published online ahead of print on 2 May 2012

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ABSTRACT 23

One of the first lines of defense against infection is the activation of the innate immune 24

system. It is becoming clear that autoimmune diseases such as rheumatoid arthritis and 25

Crohn’s disease may be caused by a disturbed innate immunity, and relating granulocyte 26

and monocyte function to patient genotype has become an important part of 27

contemporary research. Although essential to move this field forward, a systematic study 28

comparing the efficacy and suitability for functional studies of the various available 29

protocols for the isolation of these immune cells has not been performed. Here, we 30

compare human granulocyte functionality upon three enrichment protocols; 1) Ficoll 31

density gradient centrifugation, 2) anti-CD15 antibody conjugated microbeads (positive 32

selection) and 3) PolymorphoprepTM. Primary monocytes were isolated in parallel using 33

1) anti-CD14 magnetic microbeads, 2) non-monocyte depletion by antibody conjugated 34

magnetic microbeads (negative selection), 3) RosetteSepTM antibody cocktail, and 4) the 35

classical adherence protocol. Best results in terms of purity and cell functionality were 36

obtained with positive selection by magnetic microbeads for both human granulocytes 37

and monocytes. Whereas phagocytosis of E. coli bacteria was identical in all isolation 38

procedures tested, granulocyte respiratory burst was higher in positively selected cells. In 39

addition, different granulocyte enrichment procedures affect cell surface receptor 40

expression to a different extent. In toto, we propose that positive selection of granulocytes 41

and monocytes be adopted as the procedure of choice for studies on human granulocyte 42

and monocyte function, but caution investigators to be aware of possible alterations in 43

cell phenotypes upon different isolation procedures. 44

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INTRODUCTION 46

Monocytes represent 3-7 percent of total white blood cells (absolute monocyte count 1.5-47

7 ×108/ liter blood) in healthy human adults. Circulating monocytes, which are derived 48

from myelomonocytic stem cells in bone marrow, have two main functions in the 49

immune system: (1) to replenish resident macrophages and dendritic cells in peripheral 50

tissues under normal states, and (2) to patrol healthy tissues through long-range crawling 51

on the resting endothelium [10]. In response to inflammatory signals, monocytes quickly 52

move to sites of infection in the tissues, engage in phagocytosis of foreign substances, 53

and initiate an early immune response through the recruitment of neutrophils and other 54

polymorphonuclear leukocytes (PMNs). PMNs constitute the most abundant of peripheral 55

white blood cells, i.e. 40 to 60% (absolute count 25-750×108/ liter blood). Their 56

bactericidal activity is essential in the proper clearance of infectious agents, and stems 57

from their exocytosis of lysozyme and protease-containing granules, phagocytosis of 58

bacteria and the concomitant production of reactive oxygen species (ROS, respiratory 59

burst). Extravasation of PMNs to the site of inflammation in turn precedes a second wave 60

of migrating monocytes to remove rapidly apoptotic PMNs. Monocytes, macrophages 61

and dendritic cells are also capable of eliciting adaptive immune response via antigen 62

presentation, a role which has also been attributed to granulocytes in inflammatory 63

settings [6;22]. Therefore, PMN and monocytes play a pivotal role in keeping the 64

dynamic balance of human immune system [29]. 65

Interest in measuring innate immune cell functionality has substantially increased, 66

especially because of a growing acceptance of the notion that defects in innate immunity 67

contribute to the pathogenesis of autoimmune diseases. Examples of those are rheumatoid 68


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arthritis (RA), systemic lupus erythematosis (SLE), an autoimmunity against DNA, and 69

the pathogenesis of Crohn’s disease (CD), an often severe autoimmunity towards the 70

resident gut flora. CD patients exhibit a phagocyte immunodeficiency that combines a 71

primary macrophage defect and a secondary granulocytic defect [2-5;24]. Macrophages 72

from these patients demonstrate impaired cytokine secretion in response to E.coli loading 73

[24]. Furthermore, after acute trauma in the gut mucosa and skin, PMN recruitment, 74

bacterial clearance and ROS production are attenuated [20;23;25]. With respect to 75

rheumatoid arthritis, granulocytes play an important role in the induction of disease and 76

disease progression, as depletion of PMN alleviates disease [28]. One of the mechanisms 77

through which granulocytes may exacerbate disease is by the deposition of autoantibodies 78

to the joints, although relatively little is known about this phenomenon [21]. In SLE, 79

granulocytes may enhance immunological reactions by the formation of neutrophil 80

extracellular traps (NETs) which forms immune complexes consisting of DNA, 81

antimicrobial peptides and autoantibodies, which subsequently activate plasmacytoid 82

dendritic cells [13;14]. In addition, monocyte function and morphology in SLE has been 83

reported to be altered; however, phagocytosis of bacteria may be either decreased or 84

enhanced, and more studies in order to assess the real contribution of these innate 85

immunity cells have been called for [15;18]. 86

Taken together, the body of contemporary biomedical literature strongly supports 87

the concept that innate immune cell dysfunction is associated with the pathogenesis of 88

many autoimmunities [29], triggering investigations into the properties of innate immune 89

cells in patients, and comparison of their phenotype to the genotype of risk genes relevant 90

for autoimmunity. The results of such studies may well depend on the protocols 91


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employed for isolation of the immune cells. A comparison of both the yield and 92

specificity of the available protocols for monocyte and PMN isolation from patient 93

peripheral blood, as well as the relative performance of cells isolated using these 94

protocols in subsequent functional experimentation, is urgently called for. 95

These considerations prompted us to perform a systemic evaluation of the most 96

frequently used methodologies for the isolation of granulocytes and monocytes from 97

human peripheral blood. PMN were isolated in parallel using (1) Ficoll density gradient 98

centrifugation, (2) polymorphprepTM density gradient centrifugation, and (3) anti-CD15 99

antibody conjugated magnetic microbeads (positive selection), after which PMN 100

functionality was assessed by in vitro phagocytosis and ROS production assays. 101

Monocytes were isolated from peripheral blood in parallel using (1) anti-CD14 antibody 102

conjugated magnetic microbeads (positive selection), (2) non-monocyte depletion by 103

antibody conjugated magnetic microbeads (negative selection), (3) immunorosette based 104

RosetteSepTM antibody cocktail (RosetteSepTM), and (4) adherence, aiming to assess their 105

suitability for in vitro phagocytosis analysis. We conclude that positive selection of 106

granulocytes and monocytes by anti-CD15 and anti-CD14 antibody conjugated magnetic 107

microbeads, respectively, are best suited for studies in which purity is imperative, but that 108

in general the isolation method of choice should depend on the type of functional assay to 109

be used. 110

111

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MATERIALS AND METHODS 113

114

Granulocyte isolation from human peripheral blood 115

Heparin and EDTA anti-coagulated blood was obtained from healthy volunteers after 116

informed consent, and in regulation with the ethical guidelines of the institution. 117

Neutrophils were isolated as described previously [9]. In short, mononuclear cells were 118

removed by centrifugation of heparinized blood over Ficoll-Paque (Amersham), followed 119

by erythrocyte lysis with ice-cold NH4Cl solution. For positive selection, granulocytes 120

obtained from Ficoll density gradient centrifugation were subsequently subjected to anti-121

CD15 microbead isolation (Miltenyi Biotech, Amsterdam, the Netherlands), using 122

manual columns, according to the manufacturers’ instruction. Additionally, PMN were 123

isolated from EDTA anti-coagulated blood using PolymorphPrepTM (Axis-Shield, 124

Norway). When present, erythrocytes were lysed with ice-cold NH4Cl solution. Before 125

functional testing, PMN were allowed to recover for 30 minutes at 37ºC in RPMI 1640 126

supplemented with 0.5% human serum albumin (HSA; Sanquin, The Netherlands). Cells 127

were resuspended in incubation buffer (20mM HEPES, 132mM Nacl, 6mM KCl, 1mM 128

MgSO4, 1.2mM KH2PO4, 5mM glucose, 1 mM CaCl2, and 0.5% HSA) prior to subjecting 129

them to functional assays. All isolation procedures were done in parallel per healthy 130

donor. 131

132

ROS production assay 133

ROS production was performed as described [8]. Shortly, PMN (2x106 cells/ml) were 134

incubated with Dihydrorhodamine 123 (DHR 123) for 15 minutes and stimulated with 135


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1µM N-formyl-methionine-leucine-phenylalanine (fMLP) for 30 minutes. For priming 136

experiments, cells were pretreated with 5ng/ml GM-CSF for 15 minutes prior to fMLP 137

stimulation. Stimulation of PMN with fMLP was terminated by washing the cells with 138

ice-cold PBS containing 1% HSA and placing them on ice. Oxidation of DHR123 to the 139

fluorescent Rhodamine 123 was measured by FACS-analysis within 30 minutes after 140

termination of stimulation. 141

142

Monocyte isolation from human peripheral blood 143

Heparinized blood was obtained from healthy volunteers after informed consent. In order 144

to optimize monocyte isolation method, monocytes from the same healthy donor were 145

isolated by four different strategies in parallel. In short, monocyte positive selection by 146

monoclonal CD14 antibody (isotype: mouse IgG2a) conjugated microbeads (Miltenyi 147

Biotec, Germany) and monocyte negative selection by monocyte isolation kit II (Miltenyi 148

Biotec) were performed with manual columns strictly according to the manufacturer’s 149

protocol, as was monocyte isolation by RosetteSepTM (Stemcell technologies, France). 150

Monocyte isolation by adherence method was performed as described previously [7]. 151

Briefly, peripheral blood mononuclear cells were isolated immediately after collection 152

using LymphoprepTM gradients (Axis-Shield PoC As, Norway). Monocytes were further 153

enriched by virtue of their attachment to a culture plate for 2 hours, washed 3 times with 154

warm phosphate buffered saline (PBS) to remove non-adherent cells, the adherent 155

monocytes were recovered by cell scraper. The purity of monocytes was evaluated by 156

fluorescent staining with CD14-PE monoclonal antibody (UCHM1, murine IgG2a, IQ 157


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products, Netherlands) and FACS analysis. The recovery of monocytes was evaluated by 158

Trypan blue staining and counted using a Zeiss microscope. 159

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Cell culture 161

Monocytes were cultured in complete medium consisting of RPMI1640 (PAA 162

laboratories, Austria) supplemented with 10% heat-inactivated FCS (PPA) and 10 μg/ml 163

Gentamicine (Centrafarm, Netherlands) at 37°C in 5% CO2 humidified air. 164

165

Phagocytosis assay 166

GFP expression vector was transformed into E.Coli and grown in LB media until OD of 167

400 was reached. Bacteria were fixed using 4% formaldehyde. Isolated PMN were 168

resuspended (2 x106/ml) in RPMI containing 10% FCS and 1x106 bacteria were subjected 169

to PMN phagocytosis at 37°C for 15 minutes. The percentage of phagocytosing PMN 170

was determined by flow cytometry by analyzing the percentage of GFP positive PMN. 171

Phagocytosis at 0°C was used as negative control for each experiment. No FITC positive 172

PMN were observed under this condition, confirming active phagocytosis of bacteria 173

rather than attachment of bacteria to PMN membranes. Alternatively, 4% formaldehyde-174

fixed E. coli were labeled with FITC fluorescence by incubation in 1mg/ml FITC solution 175

containing 0.1M Na2CO3 pH 9.5 for 1 hour followed by complete washing. The 176

efficiency of labeling was tested by FACS analysis (see below). Bacterium concentration 177

was quantified using Quantimet HR550 image analysis software (Leica) to analyze 178

microscopic images of FITC-E. coli taken with a Leica (Wetzlar, Germany) DMRXA 179

epifluorescence microscope [30]. For monocytes, the phagocytosis assay was performed 180


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according to Mandell and Hook [19]. In short, monocytes (1× 104/ml) were incubated 181

with FITC-E. coli at a 1:5 ratio for 5 or 15 minutes at 37 °C in RPMI1640 medium 182

containing 10% heat-inactivated FCS. Thereafter, phagocytosis was evaluated 183

microscopically by counting both the number of monocytes exhibiting phagocytosis and 184

the number of bacteria phagocytosed per monocyte. At least 300 cells were counted for 185

each slide. The engulfment rather then attachment of E. coli to PMN and monocytes was 186

confirmed by confocal microscope (LSM 700, Carl Zeiss, Jena, Germany) and cultures at 187

4 oC. 188

189

FACS analysis 190

Isolated PMN or whole blood after red cell lysis were stained resuspended in 191

PBS/EDTA/1%FCS). After blocking with FcR blocking reagent from Miltenyi Biotech 192

(Amsterdam, the Netherlands) for 15 minutes, cells were labeled with anti-CD14-PerCP-193

Cy5.5 (clone M5E2), anti-CD66b-PerCP-Cy5.5 (G10F5), anti-CD15-PE (clone HI98), 194

anti-TLR2-Alexa 647 (clone TL2.1), anti-TLR4-PE (clone HTA125), anti-CD62L-195

Alexafluor647 (clone DREG56) (all from Biolegend, San Diego, CA). No FcR blocking 196

was performed when staining for FcReceptors using anti-CD16-PE (clone 3G8, 197

Biolegend), anti-CD64-PE (clone 10.1, Biolegend) or anti-CD32-FITC (clone FL18.26 198

from BD Bioscience, Franklin Lakes, NJ). After washing with PBS, fluorescence was 199

measured by flow cytometry (FACSCanto, BD Bioscience Franklin Lakes, NJ). 200

201

Analysis of purity 202


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Analysis of the purities of granulocytes and monocytes by FACS was performed using 203

PerCP-Cy5-labeled CD66b (Clone G10F5, Biolegend) and PE-labeled CD14 monoclonal 204

antibody (clone UCHM1, murine IgG2a, IQ products, Netherlands), respectively. In 205

short, cells were incubated with appropriate antibodies (10 μl per 5 × 105 cells) for 30 206

minutes on ice. Cells were washed twice in PBS/0.1% BSA before FACS analysis. For 207

identifying the platelet contaminants and observing the cytoskeleton of monocytes, 208

enriched monocytes were further stained intracellularly with TRITC conjugated 209

phalloidin (Sigma-aldrich). In short, cells were fixed in PBS/4% formaldehyde for 10 210

minutes, washed twice in PBS, permeabilized in PBS/0.1% Triton X-100 (Sigma-211

Aldrich), treated with TRITC conjugated phalloidin (25μg/ml) for 30 minutes in dark, 212

and washed three times in PBS. Platelets were identified by their morphology under 213

fluorescence microscopy. Contaminating cells in granulocyte isolates were discriminates 214

using anti-CD19-PE, clone IB19 (B-cell), anti-CD3-Amcyan clone SK7 (T-cell) and anti-215

CD14-PerCP clone M5E2 (Monocyte) antibodies. DAPI was used to exclude dead cells. 216

217

Statistics 218

For comparison of multiple isolation methods, differences between the means of the 219

groups were tested by non-parametric test for related samples (Friedman test) or unpaired 220

samples (Kruskal-Wallis). Where variance between the means of groups was found by 221

Friedman test, post-hoc analysis was performed by Wilcoxon signed Ranks testing, to 222

determine which of the groups differed from each other. Wilcoxon signed Rank non-223

parametric test for paired samples was also used for comparisons between two groups, 224


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such as the comparison of fMLP-stimulated ROS production with and without priming 225

with GM-CSF. 226

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RESULTS 228

229

Comparison of three commonly used isolation protocols for purity of enriched 230

granulocytes. 231

PMN were isolated from blood from healthy volunteers using, in parallel, three of the 232

most commonly used methods in literature. Purity of isolates was analyzed by flow 233

cytometry, and proved to be 96.2±2, 99.5±0.5 and 91.4±4.9% of nucleated cells for 234

Ficoll, anti-CD15 microbead positive selection and PolymorphprepTM isolation methods, 235

respectively (Figure 1A, Table 1). This measure, however, excludes non-nucleated cells 236

(erythrocytes and thrombocytes) as well as ghosts derived from necrotic cells. Hence, we 237

also determined the percentage of CD66b positive cells as a function of all cell-like 238

particles (i.e. all particles in which Ø >0,5 μM). When results were expressed this way, 239

Ficoll density gradient centrifugation yielded 87±3% granulocyte purity, compared to 240

88±5% for positive selection (see Figure 1B for representative example). 241

PolymorphprepTM isolation method resulted on average in 76±19% purity, largely due to 242

a higher amount of contaminating debris particles and lymphocytes (Figure 1C). Also, in 243

our hands, the highest variability in purity between isolations was observed when using 244

PolymorphprepTM, suggesting that this method is somewhat more sensitive to small day 245

to day variations. We also noticed a donor to donor variation in adherence of the 246

lymphocyte ring to the plastic disposables after centrifugation, which may account for 247

some of the contamination variability observed. When analyzing the nature of this 248

contaminating mononuclear cell fraction, these were found to be mainly CD3-positive T-249

cells, with little or no CD19+ B-cell or CD14+ monocytes (not shown). 250


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PMN are amongst the most short-lived cells in the body. They are extremely 251

fragile cells, and prone to apoptosis upon withdrawal from the blood. Importantly, 252

however, no more than 3.7% dying cells were identified by 7AAD (Fig 2D) or DAPI 253

staining (not shown) as determined by FACS analysis of any of the isolation methods 254

used, indicating that no significant cell death was induced during isolation procedures. 255

In conclusion, the best results in terms of granulocyte purity were obtained using 256

anti-CD15 microbead positive selection methods. 257

258

Comparison of granulocyte yield obtained using the three most commonly used 259

PMN isolation protocols 260

The absolute recovery rates of PMN after the three isolation procedures are shown in 261

Table 1. Typically, isolation of PMN by positive selection, though leading to the highest 262

purity, also yielded the lowest recovery rates; 9.6×105/ml peripheral blood were obtained, 263

which was significantly less then the yields obtained using either Ficoll centrifugation 264

(16×105/ml peripheral blood) or PolymorphprepTM (14.8×105/ml peripheral blood). With 265

a purity of 95.2% and the highest overall yield, granulocyte enrichment by Ficoll 266

centrifugation seems the optimal method when high numbers of cells are required. 267

268

Granulocyte functionality in a ROS production assay following different isolation 269

protocols 270

PMN are easily activated by trace amounts of bacterial lipopolysaccharides or mechanical 271

stressors. To determine whether our different isolation methods give unwanted activation 272

of PMN, we studied their ROS production. Spontaneous PMN ROS production was not 273


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different between isolation procedures (data not shown). In addition, expression of the 274

neutrophil activation marker L-selectin (CD62L), shedding of which is associated with 275

PMN activation [26], was similar between groups, comfirming that there was no bias in 276

activation of PMN between isolation methods (Figure 3A, upper left panel). 277

ROS production can be triggered by the bacterial peptide analogue fMLP, and 278

fMLP-induced ROS production is significantly enhanced in GM-CSF-primed PMN. 279

Significant differences were observed when comparing fMLP-triggered ROS production 280

between isolation methods (p=0.042 by Friedman Test). These were caused by the fact 281

that PMN isolated by PolymorphprepTM showed a lag in fMLP-induced ROS production 282

when compared to either Ficoll or positive selection methods (109±8% vs 148±18 and 283

168±28, respectively, at t=5 min, Figure 2A). Pretreatment of PMN with GM-CSF 284

resulted in a significant enhancement ROS production for all isolation methods tested. 285

However, we observed significant differences between the groups (p=0.042 by Friedman 286

Test), with PMN isolated by positive selection showing a significantly higher ROS 287

production than the other two isolation methods. These results suggest that the ability of 288

PMN to be primed for ROS production is highest in PMN isolated by positive selection, 289

which may argue for lower activation status upon isolation in these cells. Indeed, when 290

the priming of ROS production was compared between groups, significant differences 291

were observed between isolation methods (p = 0.009 by Friedman Test), with cells 292

isolated by positive selection showing a significantly higher priming capacity then PMN 293

isolated by Ficoll. Polymophprep isolation induced a higher variability in priming of ROS 294

production, but due to its lag in fMLP-induced ROS production, priming in this group 295


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was higher when compared to Ficoll-isolated PMN (figure 2B). Therefore, for ROS 296

production analysis, positive selection of PMN may be the best option. 297

298

Granulocyte functionality in an Escherichia coli phagocytosis assay following 299

different isolation protocols 300

Ficoll has been shown to change PMN shape and migratory capacity, indicating an effect 301

on cytoskeletal rearrangement [11]. Phagocytosis is dependent on the actin cytoskeleton, 302

and as such may be affected by Ficoll isolation procedures. Positive selection of 303

neutrophils relies on the antibody binding of CD15, a carbohydrate adhesion molecule. 304

Ligation of CD15 antibodies to this integrin-associated molecule may potentially affect 305

phagocytosis and adhesion of PMN [16]. We therefore studied the E. coli phagocytosing 306

capacity of PMN isolated by different methods, using phagocytosis of PMN in whole 307

blood as control. To distinguish PMN in whole blood samples, cells were stained for 308

CD15, and the percentage of PMN containing GFP-positive E.Coli was determined by 309

FACS analysis. As expected, phagocytosis of non-isolated PMN is more efficient than 310

that of isolated granulocytes, with more than 53±4% of granulocytes taking up GFP-311

positive bacteria. No significant differences in the percentage of phagocytosing cells were 312

observed in PMN isolated by Ficoll centrifugation, positive selection or 313

PolymorphprepTM (12.6±3%, 10.8±2% and 12.2±3%, respectively, n=6; p = .55, Figure 314

2C). Engulfment of bacteria rather then adhesion to PMN cell surface was confirmed by 315

confocal microscopy (supplementary movie 1). In addition, the amount of bacteria taken 316

up per cell, as determined by mean fluorescence intensity (MFI) of GFP-positive PMN, 317


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did not differ per isolation method used (MFI of 1172, 1054 and 1231 for Ficoll 318

centrifugation, positive selection or PolymorphprepTM, respectively, not shown, p=0.819). 319

320

321

Expression of PMN cell surface markers following different isolation procedures. 322

Isolation of granulocytes may affect their expression of cell surface receptors and other 323

molecules, and thereby alter specific granulocyte functions. CD15, CD66b and CD62L 324

(L-selectin) are adhesion molecules involved in PMN phagocytosis and chemotaxis. 325

Analysis of CD15 expression upon different isolation procedures reveals a slight but 326

significant decrease in CD15 and CD66b expression upon PolymorphprepTM isolation as 327

compared to Ficoll isolation and positive selection, respectively. Whereas CD62L 328

expression was comparable between groups, expression of CD66b was highest on PMN 329

isolated by positive selection, followed by Ficoll and Polymorphprep. Although this is 330

not likely to affect phagocytosis (as seen above), it is conceivable that other, untested 331

functions (e.g. migration), could potentially be affected. 332

We also assessed expression of the CD64 FcγRI, the CD32 FcγRII and the low 333

affinity FcRIII, CD16. Although CD64 expression was higher in positively selected PMN 334

as compared to PMN isolated by Ficoll, significant differences between groups were only 335

observed for CD16. PMN isolated by positive selection showed a small but significant 336

increase in CD16 expression as compared to either Ficoll or PolymorphprepTM isolates. 337

No differences in CD32 expression were observed between isolation protocols. Next, we 338

analyzed the expression of Toll like receptor (TLR) 2, TLR4 and co-receptor CD14, 339

which bind lipopolisaccharise (LPS), the major component of the outer membrane of 340


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Gram-negative bacteria such as E. coli [12]. A dramatic increase in TLR2 and TLR4 341

expression was observed on PMN isolated by positive selection. This could have 342

substantial consequences for studies into the function of these receptors as well as studies 343

into functional LPS responses, and isolation procedures should therefore be carefully 344

considered by investigators undertaking such studies. 345

346

Comparison of the four most commonly-used isolation protocols for purity of 347

enriched monocyte cultures 348

Monocytes of healthy individuals were isolated from peripheral blood in parallel by the 349

four procedures most commonly used in the literature. First we decided to assess the 350

purity of the monocyte fraction following the four different types of enrichment as a 351

percentage of all nucleated cells using FACS. Using this measure, it was observed that 352

monocytes were obtained with purities of 98.5%, 97.0%, 67.3% and 64.2% by positive 353

selection, negative selection, adherence and RosetteSepTM, respectively (Fig. 4A). 354

Furthermore, visual inspection of monocyte cultures revealed the presence of non-355

nucleated particles. When results were expressed as percentage of all recorded events (Ø 356

>0,5 μM) the monocyte purity obtained was 95.4%, 48.7%, 58.5% and 35.9% for 357

respectively positive selection, negative selection, adherence and RosetteSepTM (Fig. 4B, 358

representative example). Thus with regard to purity, positive selection yields the bests 359

results for monocyte isolation, both judged by the monocyte fraction of all nucleated cells 360

and as fraction of all cell-like bodies. Importantly, after positive selection procedures 361

during which monocytes were labeled with antibodies for CD14 receptors, abundant 362


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CD14 expression was still detected by anti-CD14 PE, indicating a functional condition of 363

these cells with respect to CD14 (Fig. 4D). 364

Next, we analyzed the nature of the contaminants in the enriched samples obtained using 365

the four different isolation procedures. As shown in Fig. 4C, compared to samples 366

obtained through positive selection, large amounts of platelets remained in the enriched 367

monocyte cultures after negative selection. After adherent isolation procedure, the major 368

contaminants are lymphocytes and platelets. After RosetteSepTM procedures, we observed 369

large amounts of lymphocytes, platelets, and non-specific cellular aggregates, which 370

significantly compromise the purity of this monocyte culture. 371

372

Comparison of monocyte yield obtained using the four most commonly-used 373

monocyte isolation protocols 374

The absolute recovery rates of monocytes after the four isolation procedures are depicted 375

in Table 2. The purities and recoveries of monocytes after positive selection, negative 376

selection, and RosetteSepTM procedures were highly reproducible. During the enrichment 377

procedure by adherence, due to the difficulties to standardize the washing steps by which 378

monocytes and lymphocytes are separated, outcomes were more variable, at least in our 379

hands. In this respect, we observed that this methodology is sensitive to inadequate 380

washing of cultures resulting in significantly compromised monocyte purity, whereas 381

excess washing has substantial consequences with respect to monocyte recovery yield 382

(not shown). Nevertheless, typically, isolation by adherence enriched monocytes to a 383

purity of 68.1% and a recovery of 2.0×105/ml peripheral blood, which is not markedly 384

different from the yields obtained using either positive or negative selection. The 385


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RosetteSepTM procedure leads to substantially better results in this respect and takes less 386

time, but the cultures obtained suffer from impurities (see above) and on a per monocyte 387

basis, it remains slightly more expensive (Table 2). Viability of monocytes in all isolates 388

was >99%, as determined by fluorescence microscopy of DAPI stained nuclei (for 389

example, see Fig. 4D). 390

391

Monocyte functionality in a Escherichia coli phagocytosis assay following different 392

monocyte isolation protocols 393

Along with TLR4, CD14 acts as a co-receptor on monocytes, detecting bacterial LPS. In 394

order to evaluate the effect of CD14 antibody binding during positive selection 395

procedures on phagocytic capacity of enriched monocytes, we challenged monocytes 396

enriched by positive selection or negative selection with FITC-labeled E. coli. 397

Phagocytosis index is significantly influenced by bacteria/monocyte ratio [17]. For the 398

studies aiming to compare phagocytic capacity of monocytes, cells were challenged with 399

a bacteria/monocytes ratio of 5:1 for 5 minutes to induce uptake of E.Coli in 50%-80% of 400

all monocytes, an optimal set-up to show the maximal differences between samples. 401

Phagocytosis index was calculated as the percentage of monocytes exhibiting E. coli 402

ingestion as determined by fluorescence microscopy. Importantly, E. coli phagocytosis 403

of monocytes does not show significant differences, regardless of the isolation procedure 404

used (although positive selection was slightly superior (n=3, p>0.05) (Fig. 5A). Culturing 405

monocytes isolated by positive selection for 16 hrs instead of 2 hrs prior to phagocytosis 406

assay had no impact on E. coli phagocytic capacity, nor did the time of E.Coli challenge 407

(a 2:1 bacteria/monocytes ratio was employed to allow for a longer time of E. coli 408


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challenging without risking monocyte saturation) (Fig. 5B). The engulfment of E.coli by 409

monocytes rather than attachment to the cell surface was confirmed by confocal 410

microscopy (Fig. 5C and Supplementary movie 2). Importantly, we also observed active 411

phagocytosis of platelets and ghosts (Supplementary Fig. 1), potentially suggesting 412

competing effects between E. coli and platelets and stressing the importance of an 413

isolation procedure that does not yield such contaminants in the culture. Therefore, in 414

toto, the anti-CD14 antibody-conjugated magnetic microbeads (positive selection) 415

protocol appears the best suited monocyte isolation method for this type of phagocytosis 416

analysis. 417

418


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DISCUSSION 419

Although some studies into cell-populations may be performed on whole blood, thus 420

preventing the need for potentially cell-activating isolation procedures, for many studies, 421

including those on the involvement of granulocytes or monocytes in the pathogenesis of 422

autoimmune diseases, isolation of a pure population of cells is essential. Although all four 423

monocyte isolation procedures tested in this study yielded functional phagocytosis-424

competent monocytes, results with respect to purity and recovery are markedly different 425

for the different procedures. We view especially purity as a concern, as this may be 426

important for functional studies. We observed when challenging monocyte cultures with 427

FITC-labeled E. coli, that contaminating lymphocytes markedly compromise the 428

accuracy of phagocytosis quantification. This was due to two reasons: first, as a result of 429

the extremely similar morphology of monocytes and lymphocytes, quantifying the 430

percentage of phagocytosing monocytes by fluorescence microscopy is markedly 431

disturbed by lymphocyte contamination. Second, we observed that lymphocytes are also 432

able to adhere E. coli to their cell surface (data not shown), which may further hamper 433

quantification. This is especially obvious when monocytes are mixed with a large 434

population of lymphocytes (eg. PBMC) or a large amount of bacteria is loaded. 435

Furthermore, we observe that co-purified thrombocytes and cell ghosts are also subject to 436

phagocytosis, with unknown effects on monocyte physiology and thus possibly 437

compromising experimentation. In addition, it has been shown that isolating a pure cell 438

population is important for other functional studies of monocytes, e.g. antigen 439

presentation and cytokine production. For these reasons, isolation of monocytes 440

employing positive selection and anti-CD14 conjugated microbeads appears the 441


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technology of choice, at least for phagocytosis analysis. However, due to the high costs of 442

commercial reagents and required instruments for positive selection, in studies where the 443

purity of monocytes is not strictly required, traditional adherent isolation procedures 444

remain an option. For instance, in studies using monocyte-derived dendritic cells, the 445

lymphocyte contaminants may be less important as the latter will not survive for longer 446

than 1 week in the absence of IL2. 447

With respect to the absolute yields obtained, the RosetteSepTM comes out 448

superior, but does not yield pure cultures. The other technologies are comparable, 449

including the traditional adherence protocol, the latter is subject to substantial 450

experimentator- and day-to-day-dependent variability, hampering its application and 451

comparison of results. Negative selection yields less pure cultures and is more expensive 452

per monocyte and thus appears to be a less attractive choice. 453

Regarding PMN isolation, comparison of three widely used methods showed that 454

isolation by positive selection yielded isolates with the highest purity, followed closely by 455

Ficoll centrifugation. PolymorphprepTM isolates showed the highest impurities, which can 456

be problematic in certain experiments. Another considerable drawback in the use of 457

PolymorphprepTM is that relatively high purities can only be reached when using EDTA 458

as anticoagulation agent, which may not always be practical. Theoretically, the main 459

advantage of PolymorphprepTM is the elimination of erythrocyte lysis steps. However, red 460

blood cell contamination of the PMN ring is often observed (up to 6% of isolate, as per 461

manufacturer’s datasheet), and a mild yet potentially cell-activating lysis step may 462

therefore nonetheless be required. 463


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Isolates from positive selection methods showed the highest ROS production in 464

response to GM-CSF + fMLP treatment, which may imply that this method of isolation 465

induces the least desensitization of PMN. Hence, positive selection seems the method of 466

choice for studying ROS production, closely followed by Ficoll isolation. ROS 467

production is dependent on the actin cytoskeleton, in that disruption of actin 468

polymerization results in increased ROS levels [25]. As CD15 ligation may affect 469

cytoskeletal rearrangement, it is theoretically possible that positive selection using CD15 470

antibodies can enhance ROS production or affect phagocytosis. However, phagocytosis 471

of E. Coli was similar in all three methods tested, and fMLP-induced ROS production in 472

positively selected isolates was equal to that in isolates from Ficoll centrifugation, 473

indicating that CD15 engagement in positive selection procedures at least does not affect 474

these functions. Although no differences in the PMN activation marker CD62L were 475

observed, our results did clearly show an upregulation of the toll like receptors TLR2 and 476

TLR4 on PMN isolated by positive selection. Although this is not likely to affect either 477

the ROS production or E.Coli phagocytosis tested here, this increase may nevertheless 478

influence other cellular assays investigators into innate immunity may want to study. 479

Careful consideration of different neutrophil isolation techniques is therefore required for 480

every functional study considered by investigators, and may depend on the type of assay 481

desired. 482

In this study, we have compared 4 widely used monocyte isolation 483

procedures, and 3 commonly used granulocyte isolation methods. However, other 484

isolation procedures have been described that were not covered in this study. For 485

instance, monocytes may be isolated by CD14-positive selection in the magnet-based 486


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MagCellect system from R&D Systems. However, this company does not at this time 487

offer granulocyte isolation kits. In addition, using Elutra™ separator, isolates by which 488

monocytes are enriched on the basis of size and to a lesser extent density from an entire 489

apheresis product have been described to result in approximately 75% purity of 490

monocytes. However, this procedure requires an automated system, and may therefore 491

not be suitable for every laboratory. For granulocytes, EasySep® magnetic isolation kits 492

based on CD66 expression are available from Stemcell technologies. How these different 493

isolation procedures (CD15 versus CD66b positive selection) affect PMN cell function 494

remains to be investigated. 495

In conclusion, if high purity of cells is required, and limited cells are needed, we 496

would consider that PMN and monocyte isolation using positive selection is the most 497

suitable method. Where high purity of PMN or monocytes is not strictly required, Ficoll 498

density gradient centrifugation and the traditional adherent isolation procedure remain 499

good options for PMN and monocytes purification, respectively. However, investigators 500

into innate cell functions should be well aware of possible alterations in cell phenotypes 501

upon different isolation procedures, and the best isolation procedure may depend on the 502

assays planned to be used. 503

504


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Acknowledgement: G.M.F. was supported by the Dutch Cancer Society (grant EMCR 505

2010-4737). 506

507


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FIGURE LEGENDS 508

Figure 1. The purities of enriched granulocytes. (A) Representative flow cytometry 509

histograms (of n=4) showing the purity of enriched granulocyte isolates, obtained by 510

either 1) Ficoll density centrifugation, 2) anti-CD15 antibody conjugated magnetic 511

microbeads (positive selection), 3) Density centrifugation using PolymorphprepTM. 512

Histograms represent CD66b positive cells after gating for nucleated cells using forward 513

and sidescatter profiles. (B) The purities of granulocytes after isolation are expressed as 514

fraction of all cell-like particles (including nucleated cells, non-nucleated cells, and 515

ghosts) after enrichment. (C) Forward and side scatter plots of the enriched samples 516

showing the nature of contaminants as identified using traditional forward and side 517

scatter morphology. 518

519


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Figure 2. Functional testing of enriched granulocyte fractions. (A) Granulocytes 520

isolated by Ficoll density centrifugation, anti-CD15 antibody conjugated magnetic 521

microbeads (positive selection) or density centrifugation using PolymorphprepTM were 522

subjected to ROS production analysis. Stimulation with fMLP results in low production 523

of ROS, indicative of resting cells, whereas priming of cells with GM-CSF yields the 524

highest fMLP-induced ROS production in granulocytes isolated by CD15- microbeads 525

positive selection. Mean ± SEM of 6 experiments is shown. Asterisks indicate 526

significantly higher ROS production in GM-CSF+fMLP stimulated cells as compared to 527

fMLP alone (P<0.05). (B) Priming capacity was determined by expressing ROS 528

production in GM-CSF cells as a percentage of ROS production in unprimed, fMLP-529

stimulated granulocytes (Mean ± SEM of 6 experiments). (C) Enriched granulocytes 530

were challenged with FITC-expressing E.Coli for 15 min, after which FITC fluorescence 531

of isolates was determined by FACS analysis. Whole blood was used as positive control 532

(Mean ± SEM of 6 experiments). (D) Cell viability of enriched PMN was determined by 533

measuring the percentage of 7AAD positive PMN by flow cytometry. (Mean ± SEM of 6 534

experiments) 535

536


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Figure 3. Expression of cell surface markers on isolated granulocytes. 537

Granulocytes isolated by Ficoll density centrifugation, anti-CD15 antibody conjugated 538

magnetic microbeads (positive selection) or density centrifugation using 539

PolymorphprepTM were subsequently stained for CD62L, CD15, CD66b, CD64, CD16, 540

CD32, CD14, TLR2 and TLR4. Except for 7AAD staining, which was present in less 541

then 5% of PMN, all other markers were present on >98% of all isolated cells, and the 542

median fluorescence intensities of these markers was determined. Mean ± SEM of 6 543

experiments is shown. 544

545


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Figure 4. The purities of enriched monocytes. (A) Representative flow cytometry 546

histrograms (of n=3) showing the purity of enriched monocyte cultures, obtained by 547

either 1) anti-CD14 antibody conjugated magnetic microbeads (positive selection), (2) 548

non-monocyte depletion by antibody conjugated magnetic microbeads (negative 549

selection), (3) a classical adherence protocol, and (4) immunorosette based RosetteSepTM 550

antibody cocktail, as percentage of monocytes out of nucleated cells. (B) The purities of 551

monocytes after isolation are expressed as fraction of all cell-like particles (including 552

nucleated cells, non-nucleated cells, and ghosts) after enrichment. (C) Analysis of the 553

nature of contaminants in the enriched samples as identified using traditional forward and 554

side scatter methodology. (D) Monocytes purified by positive selection were stained for 555

CD14 and nuclei using anti-CD14 PE antibody (red) and DAPI (blue). 556

557


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Figure 5. Phagocytic capacity of monocytes. (A) Monocytes obtained through the 558

positive selection and negative selection procedures were challenged with FITC-labeled 559

E. coli for 5 minutes, in parallel. Phagocytosis of E. coli is expressed as the percentage of 560

monocytes displaying phagocytosis. E. coli phagocytosis of monocytes after the two 561

isolation procedures did not show significant difference, although the level of 562

phagocytosis in negative selection group is slightly lower (n=3, p>0.05, values were 563

presented with Mean±SEM) (B) After positive selection procedure, monocytes after 16 564

hour culture exhibit the same E. coli phagocytotic capacity compared to the 2 hour 565

culture (representative of 3 independent experiments). (C) A representative trans-566

sectional image of monocytes by confocal microscope confirming that E. coli are 567

engulfed rather by attached to monocytes, red: cytoskeleton of monocytes (F-actin) 568

stained by TRITC-phalloidin; green: FITC-labed E.coli. 569

570


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Table 1. Comparison of the yield and purity of granulocytes, time consumption and costs of

the three different isolation procedures evaluated in the present study.

Methods Yield Purity Procedure time Cost(€/10ml blood)

Ficoll centrifugation 16±6.3×105 96.2±1.7% 1.5 hours 5.4

Positive selection 9.6±7.9×105 99.5±0.5% 2.5 hours 33*

PolymorphprepTM 14.8±4.3×105 91.4±4.9% 1 hour 6.1

Note: The cost for positive selection did not include the magnetic separator. Purity shown is

of total nucleated cells. *Cost of positive selection depends on the yield of PMN after Ficoll

centrifugation, and shown is the amount calculated based on 107 total cells. Mean ± SD is

shown of 6 independent experiments. Yield of PMN isolation differed significantly between

groups (P = 0.04 by Friedman Test). Specifically, Positive selection yielded lower numbers of

PMN compared to either Ficoll or Polymorphprep (P = 0.028 and 0.046, respectively. Purity

of PMN differed significantly between groups (P = 0.009 by Friedman Test). Post-hoc

analysis revealed that purity of PMN isolated by Polymorphprep was significantly lower than

per Ficoll centrifugation or Positive selection (P = 0.03 and 0.03, respectively).


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Table 2. Comparison of the yield and purity of monocytes, time consumption and costs of the

four different isolation procedures evaluated in the present study.

Methods Yield Purity Procedure time Cost (€/10 ml blood)

Positive selection 2.2±0.3×105 98.5±0.4% 2.5 hours 23

Negative selection 2.2±0.3×105 97±0.7% 3 hours 26

Adherence 1.9±0.6×105 67.3±3.6% 4 hours 8

RosetteSep® 4.2±0.6×105 64.2±4.3% 1.5 hours 46

Note: The cost for positive selection and negative selection did not include the magnetic

separator. The yield of monocytes enriched by adherence was shown for the optimal washing

condition. Purity shown is of total nucleated cells. Mean of 5 experiments ± SD is shown. The

yield and purity of monocytes were significantly different among procedures (P = 0.009 and

P = 0.001, respectively by Kruskal-Wallis test).


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