Mass Cytometry Enables Absolute and Fast Quantification of Silver

Subscriber access provided by Nottingham Trent University

Technical Note

Mass Cytometry Enables Absolute and Fast Quantification of Silver Nanoparticle Uptake at the Single Cell Level Ana Lopez Serrano Oliver, Andrea Haase, Anette Peddinghaus, Doreen Wittke, Norbert Jakubowski, Andreas Luch, Andreas Grützkau, and Sabine Baumgart Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01870 • Publication Date (Web): 20 Aug 2019 Downloaded from pubs.acs.org on August 21, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

1

Mass Cytometry Enables Absolute and Fast

2

Quantification of Silver Nanoparticle Uptake at

3

the Single Cell Level

4

Ana Lopez-Serrano Olivera, Andrea Haaseb, Anette Peddinghausc, Doreen Wittkeb, Norbert

5

Jakubowskia,d, Andreas Luchb, Andreas Grützkauc, Sabine Baumgartc*

6 7 8 9 10

a Bundesanstalt

für Materialforschung und -prüfung (BAM)- Richard-Willstaetter-Str. 11, 12489, Berlin, Germany; bGerman Federal Institute for Risk Assessment (BfR), Department of Chemical and Product Safety, Max-Dohrn-Str. 8-10, 10589, Berlin Germany; cDeutsches RheumaForschungszentrum Berlin (DRFZ), ein Leibniz Institut, Charitéplatz 1, 10117, Berlin, Germany; dSpetec GmbH, Berghamer Str. 2, 85435 Erding, Germany.

11 12

KEYWORDS: silver nanoparticles, differentiated THP-1 cells, macrophages, absolute

13

quantification, mass cytometry

14 15 16 17

*

Address correspondence to [email protected].

ACS Paragon Plus Environment

1

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 17

1

ABSTRACT

2

In the last decades, significant efforts have been made to investigate possible cytotoxic effects of

3

metallic nanoparticles (NPs). Methodologies enabling precise information regarding uptake and

4

intracellular distribution of NPs at the single cell level remain to be established. Mass cytometry

5

(MC) has been developed for high-dimensional single cell analyses and is a promising tool to

6

quantify NP-cell interactions. Here, we aim to establish a new MC-based quantification procedure

7

to receive absolute numbers of NPs per single cell by using a calibration that considers the specific

8

transmission efficiency (TE) of suspended NPs. The current MC-quantification strategy accept TE

9

values of complementary metal solutions. In this study, we demonstrate the different transmission

10

behavior of 50 nm silver NPs (AgNP) and silver nitrate solution. We have used identical AgNPs

11

for calibration as for in vitro-differentiated macrophages (THP-1 cell line) in a time- and dose-

12

dependent manner. Our quantification relies on silver intensities measuring AgNPs in the same

13

detection mode as the cells. Results were comparable with the TE quantification strategy using

14

AgNPs but differed when using ionic silver. Furthermore, intact and digested cell aliquots were

15

measured to investigate the impact of MC sample processing on the amount of AgNPs/cell.

16

Taken together, we have provided a MC-specific calibration procedure to precisely calculate

17

absolute numbers of NPs per single cell. Combined with its unique feature of multiplexing up to

18

50 parameters, MC provides much more information on single cell level than single cell

19

inductively coupled plasma mass spectrometry (SC-ICP-MS) and therefore, offers new

20

opportunities in nanotoxicology.


2

Page 3 of 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2

INTRODUCTION

3

In recent years, the number of products containing inorganic nanoparticles (NPs) has globally

4

increased due to their unique chemical and physical properties.1,2 For NP exposure risk assessment

5

methods are needed to study NP-cell interactions providing not only qualitative but also

6

quantitative data.3 Recently, the slice and view technology of fast ion bombardment (FIB)

7

combined with the secondary electron microscopy (SEM) technique was used to quantify 75

8

nm silver NPs (AgNPs) in a single cell, but has limited high-throughput capacities.4

9

Inductively coupled plasma mass spectrometry (ICP-MS), traditionally a bulk

10

measurement of lysed NP-exposed cells that provides averaged data5,6, but operated in

11

“single particle” detection mode enables this methodology to enter the field of single-cell

12

analysis (SC-ICP-MS).7,8 Although ICP-MS covers a wide mass range from 7 to 250 atomic

13

mass units (amu), SC-ICP-MS with a quadrupole or sector-field mass analyzer is limited

14

to record only one isotope in the short duration of a cell event.9 Therefore, SC-ICP-MS

15

alone does not allow multiparametric analyses at the single cell level, which are usually

16

necessary in many fields of basic and medical research. This gap was closed by the

17

introduction of the ICP-time of flight (TOF)-MS-based mass cytometry (MC) in 2009,

18

which enabled a simultaneously detection of multiple elements per cell event.10 The

19

characterization of single cells is ensured by the combination of many different antibodies

20

conjugated with heavy metal ions in actual devices covering the mass range between 75 and 209

21

amu (Helios)11 (Table S-1 (ESI)). Recently, its application has expanded to the absolute

22

quantification of inorganic metal-NPs associated with single cells using dissolved metal solutions

23

as calibrants similar to approaches by conventional ICP-MS.12,13 However, calibration strategies

24

need to be carefully chosen because of varying transmission efficiencies (TE) of elements due to

25

their ionization characteristic within the plasma, their physical state and possible matrix effects.14

26

In this study, we describe TE differences of ionic and particulate silver by MC. The phagocytic

27

THP-1 cell line served as in vitro model15 to demonstrate the improvement of the absolute


3


Page 4 of 17

1

quantification of AgNPs per single-cell when AgNPs were used for calibration instead of applying

2

a calculation based on the TE of dissolved silver nitrate (AgNO3) as it was reported by Ivask et

3

al.13 Results received from intact cells were compared to digested cells. NP detection was

4

accompanied by the detection of macrophage-specific immunophenotypic markers.

5


4

Page 5 of 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2

RESULTS AND DISCUSSION

3

Transmission efficiency of silver in different physical states. When a ‘CyTOF’ mass cytometer

4

operates in the ‘liquid mode’, a constant signal from ions (ion counts) derived from a

5

usually used metal solution of a selected mass channel is integrated over time. To

6

determine the TE of ionic and particulate silver according to Tricot et al

7

increasing Ag concentrations of ionic standard solutions and AgNP aqueous suspensions were

8

measured in ‘liquid mode’. As shown in Figure 1, different slopes of both linear regression curves

9

result in different TE of 1.04x10-5 and 0.56x10-5 for dissolved AgNO3 and AgNPs, respectively,

10

indicating a difference in the nebulization efficiency, ionization behavior and overall transmission

11

of silver ions on their way to the detector as it was previously described.14

16

the

107Ag

signal of

12 13

Figure 1: Linear regression including three technical replicates with their standard deviations of

14

AgNO3 standard solutions and AgNPs suspensions obtained via MC in ‘liquid mode’. The graph

15

shows

16

(AgNO3 was corrected to ionic silver).

107Ag

intensities expressed as ion counts of different Ag concentrations in µg Ag L-1


5


Page 6 of 17

1

Calibration with AgNPs suspensions in ‘cell mode’. Single cells or metal-NPs can be detected

2

in ‘cell mode ’ by triggering on a strong signal derived e.g. from DNA staining or from the NP

3

metal core. Here, the transient ion signal is integrated over a single event.10,11 To quantify

4

the AgNP uptake of THP-1 cells at the single cell level, a similar external calibration as previously

5

described9 was performed by measuring AgNP suspensions with increasing Ag concentrations. As

6

indicated in Table S-2, proportional to the AgNP concentration, the number of particle events

7

exhibits a linear increase (R2=0.99615). The

8

independent of the concentration. Therefore, an average of

9

(arithmetic mean/median) corresponds to a single 50 nm AgNP with 0.743 fg (femtogram) Ag per

107Ag

signal intensity per AgNP was constant and 107Ag

intensity of 7.9/7.2 ion counts

10

NP specified by the manufacturer.

11

Cell triggering strategy. In MC, a DNA intercalator commonly used as cell identifier allows

12

discriminating between intact nucleated cells, cell fragments and non-cell associated events. To

13

stain the nuclei, permeabilization of the cell membrane is required. Results revealed no significant

14

difference in the silver intensity when cells were treated with or without an DNA intercalator,

15

confirming that the AgNPs remained stably internalized in the cells (Figure S-1A and S-1B) as

16

previously reported by transmission electron microscopy (TEM).4 Furthermore, the cell viability

17

marker cisplatin10 and the pan leukocyte marker CD45 allowed a pre-gating on single live

18

nucleated leukocytes as illustrated in Figure S-2. Differentiation-dependent heterogeneities of

19

THP-1 cells were monitored by the detection of surface markers such as CD36 and CD11c (2-

20

integrin alpha X).17 Since CD36 expression was induced by PMA with a different kinetic

21

than CD11c (Figure S-3, (ESI)), differentiated THP-1 cells were subdivided into two subsets,

22

both expressing CD11c at comparable extents but differing in the expression of CD36 (Q1 as

23

CD11+CD36+ and Q2 as CD11+CD36dim) (Figure 2A).

24

Quantification of AgNP uptake per single cell. THP-1 macrophages were exposed to two

25

different doses of AgNPs (0.1 and 1 mg L-1) for 4 and 24 h and analyzed in the ‘cell mode’

26

compared to cells not exposed to AgNPs. Untreated controls were used to determine a signal-

27

intensity threshold using the 95th percentile of 107Ag intensity. In this study, the background noise

28

possibly caused by impure reagents, buffers and water was negligible (median107Ag ~ 2, 95th

29

percentile107Ag ~ 15, standard deviation ~ 11 ion counts) since all median107Ag values in AgNPs


6

Page 7 of 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

treated samples listed in table S-3 were at least three times higher than the standard deviations95th

2

percentile107Ag

3

(Figure 2A). The histograms for the Ag signal intensities show a broad distribution indicating a

4

high heterogeneity within the single cells regarding their capacity to take up the NPs. A more

5

homogeneous distribution of AgNP-uptake could be observed after 24 h exposure (Figure 2A)

6

indicating a saturation of silver uptake. We further calculated the absolute uptake of AgNPs/cell

7

based on the median107Ag values of CD36+ and CD36dim THP-1 subsets (Q1/Q2) and an average

8

median107Ag of 27.6 ion conts for a single 50 nm AgNP (Figure 2B, Table S-3). Although

9

phenotypically different, similar kinetics in AgNP-uptake were observed for both subsets. The

10

absolute amount of AgNPs per cell was time- and dose-dependent and ranged from 4 to 75, i.e. 3

11

to 56 fg Ag/cell. These results are in accordance with those ones reported for less maturated, non-

12

adherent THP-1 cells measured by SC-ICP-MS.9

of the control samples. After 4 h exposure, AgNPs were detectable in almost every cell

13

14


7


Page 8 of 17

1

Figure 2: Differentiated THP-1 cells were exposed to AgNPs for 4 h (left) and 24 h (right) at

2

concentrations of 0.1 and 1 mg L-1 in comparison to non-treated cells. In the figure 2A two-

3

dimensional dot-plots for CD11c and CD36 show two subsets differing in the expression of CD36

4

when pre-gated on single live CD45+ cells (191Ir/193Ir+, 195Pt-,140Ce-, CD45-154Sm+ cells), marked

5

as Q1 and Q2. The

6

adjusted to identical CD45+ counts. Figure 2B depicts the absolute numbers of AgNPs per cell

7

including their standard deviations of three independent technical replicates.

8

Comparison of calibration strategies based on AgNPs or ionic silver. Quantitative results

9

obtained from measurements in ‘cell mode’ were compared to values calculated with the resulting

10

TE from ionic silver standards measured in ‘liquid mode’ according to Ivask et. al.13 In comparison

11

to the results obtained in ‘ cell mode ’ , diminished values were obtained with the TE-based

12

calculation (difference of 60%). Thus, in general, MC allows the usage of identical NPs for

13

toxicological experiments and for calibration. In contrast, the TE-based calculation relies on the

14

availability of complementary standard solutions.

15

Validation of single cell quantitative results. Data retrieved from NPs calibration were compared

16

with data obtained from digested cell aliquots taken before and after antibody staining to address

17

the impact of cell processing procedure. Samples were measured in ‘liquid mode’ and calibration

18

was performed with ionic silver standard solutions. Significant differences in AgNP uptake per

19

cell were observed when comparing samples measured in ‘cell mode’ (including cell and non-cell

20

associated events, Figure S-4) and digested samples before antibody staining, while in digested

21

aliquots after sample processing similar values were obtained (Figure 3, Table S-4) concluding

22

that during cell preparation (e.g. antibody incubation, overnight fixation) cell membrane became

23

permeable causing a loss of NPs that are not completely compartmentalized in endosomes.

24

While similar numbers of AgNPs/cell after 4 h AgNP exposure were observed when comparing

25

results obtained from digested cells taken after cell processing measured in ‘liquid mode’ and

26

results from samples measured in ‘cell mode’ (considering the contribution of free AgNPs and cell

27

associated AgNPs), after 24 h AgNP exposure, digested cells showed two times higher values

28

reaching >100 AgNPs per cell or >92 fg Ag per cell, respectively (Figure 3, Table S-3). Related

29

plots from samples measured in the ‘ cell mode ’ revealed a high intensity for the

30

(Figure S-3), indicating the maximal number of AgNPs that can be taken up by THP-1 cells or that

107Ag

intensities are illustrated in histograms. Samples were normalized and

107Ag

signal


8

Page 9 of 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

can be detected by the CyTOF v1 instrument. Yang et al.12 estimated 343 gold particles per cell

2

with a diameter of 50 nm as a maximum before saturation of the detector on a CyTOFv2 or Helios

3

instrument. This and the observation of a sharp decline of the

4

Figure 2 at the highest AgNPs dose and exposure time might indicate a partial saturation of the

5

detector when approximately 100 AgNPs per cell were reached since the CyTOFv1 instrument has

6

a one magnitude lower dynamic range than the Helios instrument.

107Ag

signal in the histogram of

7 8

Figure 3. Quantitative analysis of AgNPs taken up by differentiated THP-1 cells exposed to 0.1

9

and 1.0 mg L-1 AgNPs for 4 and 24 h, respectively. Samples were either detected in ‘cell’ or ‘

10

liquid mode’. The results are shown for a representative single biological experiment measured in

11

three replicates.

12


9


Page 10 of 17

1

CONCLUSIONS

2

We could demonstrate that the TE of silver present in different physical states differs tremendously

3

when detected by MC. Consequently, for an absolute quantification of metallic NPs at the single-

4

cell level, we recommend to use ideally reference NPs as calibrators instead of metal standard

5

solutions and to measure them like cells in the ‘ cell mode ’ . So, the here described MC-based

6

quantification combines the best out of the analytical ICP-MS and the single cell cytometry world

7

and offers new options for nanotoxicological analyses of NP-cell interactions in cell lines and in

8

complex biological samples such as blood or other body fluids. However, complementary

9

methodologies need to be considered to distinguish between binding and intracellular uptake of

10

NP, to monitor NPs out of the mass range of mass cytometers and their possible

11

transformation/degradation within the endocytic pathway of cells.

12 13 14


10

Page 11 of 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

MATERIALS AND METHODS

2

Chemicals and Reagents. AgNPs (51 ± 6 nm diameter, Citrate BioPure Silver) were purchased

3

from nanoComposix (San Diego, CA, USA). Phosphate Buffered Saline (PBS) was prepared from

4

10x PBS (Biomol GmbH, Hamburg, Germany, adjusted to pH 7.4). Silver standard solutions of

5

dissolved silver nitrate (AgNO3), phorbol-12-myristate-13-acetate (PMA), accutase, 65% w/w

6

ultrapure grade nitric acid and hydrogen peroxide were purchased from Merck (Darmstadt,

7

Germany). Fetal calf serum (FCS), Roswell Park Memorial Institute media (RPMI 1640), 4-(2-

8

hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pyruvate, penicillin, streptomycin and

9

L-glutamine were provided by Pan Biotech GmbH (Aidenbach, Germany). A live/dead fixable

10

near-IR dead cell kit was purchased by Thermo Fisher Scientific (Waltham, MA, USA). Four

11

element EQ calibration beads, iridium intercalator and anti-human CD45-154Sm (HI30) was

12

obtained from Fludigm Corp (South San Francisco, CA, USA). The metal-labeled antibodies anti-

13

human CD36-166Er (AC160), and CD11c-160Gd (Mj427G12) were conjugated by using the

14

MAXPAR labeling kit (Fluidigm) as described.18 Cis-Platinum (II)-diamine dichloride (cisplatin)

15

was obtained from Enzo Life Sciences GmbH (Lörrach, Germany). Ultrapure (18.2 MΩ cm-1)

16

from Milli-Q water purification system (Darmstadt, Germany) was used throughout the study.

17

Cell Culture and Differentiation of THP-1 cells. The THP-1 cell line obtained from the German

18

Collection of Microorganisms and Cell Cultures GmbH (DSMZ, Braunschweig, Germany) was

19

maintained in RPMI 1640 media supplemented with 10% FCS, 10 mM HEPES, 1 mM pyruvate,

20

100 U m L-1 penicillin, 100 µg m L-1 streptomycin and 2 mmol L-1 L-glutamine. THP-1 cells (1x107

21

mL-1) were differentiated using PMA at a final concentration of 100 ng mL-1 for 48 h at 37 °C in

22

5% CO2 in air. Finally, cells were washed three times with PBS.

23

Exposure of Differentiated THP-1 Cells to AgNPs. The AgNPs (50 nm) suspension at a stock

24

concentration of 1.04 g L-1 (correlates to 1.4 x 1012 NPs mL-1) was sonicated for two min to avoid

25

AgNP aggregation. Differentiated THP-1 cells were exposed to AgNPs at final concentrations of

26

0.1 and 1.0 mg L-1 for 4 and 24 h. After incubation, cells were rinsed and washed three times with

27

PBS to remove excessive AgNPs.

28

Adherent cells were enzymatically detached from the surface of the cell culture bottle by

29

incubating with 5 mL accutase for 10 min at 37 °C in 5 % CO2 and washed with RPMI 1640 media,

30

centrifuged and further washed three times with PBS. Cell aliquots were subjected to antibody

31

staining or digestion followed by mass cytometric measurement.


11


Page 12 of 17

1

Surface Marker Staining for Mass Cytometric Measurement. After AgNPs exposure, cell

2

viability was monitored by using a live/dead fixable near-IR dead cell stain kit and measured on a

3

fluorescence-activated cell scanning Aria III flow cytometer (Becton Dickinsons, Franklin Lakes,

4

NJ, USA) (Table S-5). Cell sample processing for CyTOF measurement was described before.18

5

Briefly, cells were stained with cis-platinum followed by antibody cocktail staining including anti-

6

human CD45-154Sm, CD36-166Er, and CD11c-160Gd. After cell fixation with 4% para-

7

formaldehyde overnight, DNA was stained with 191/193Ir intercalator, cells were washed with PBS

8

and suspended in Milli-Q water. Cells were counted using a MACSQuant analyzer (Miltenyi

9

Biotec GmbH, Bergisch Gladbach, Germany) and adjusted to 5 x 105 cells mL-1.

10

Digestion of Cells for Mass Cytometric Measurement. An aliquot containing 1x105 THP-1 cells

11

was digested overnight at 4°C by adding 0.15 mL 65% nitric acid. Next day, 0.05 mL hydrogen

12

peroxide were added, and samples were diluted with Milli Q water until a final volume of 10 mL

13

for their measurement in ‘liquid mode’.

14

Mass Cytometer Set Up and Data Acquisition/Processing. A CyTOFv1/v1.5 (Fluidigm)

15

instrument was set up as described before.19 Analytes were injected via a 450 µL sample loop by

16

flow injection analysis using Millipore Q water as carrier at a flow rate of 0.045 mL min-1. Cells,

17

beads and NPs were acquired in the instrument dual-calibration mode, with noise reduction turned

18

on (‘cell mode’) using CyTOF instrument control software v5.1.6.4.8 and upgraded to v6.0.6.2.6.

19

The event length was set from 10 to 75 for cell suspensions and from 1 to 75 for AgNPs as

20

illustrated in Figure S-5. Four element EQ Beads were added as internal standard to correct time-

21

dependent variations in the instrument.11 Exported FCS data files were normalized (Helios

22

software v6.0.6.2.6). Data were manually analyzed on visual inspection using FlowJo software

23

v10 (TreeStar, Ashland, OR, USA). For all dot plots and calculations only

24

considered.

25

To determine possible spillover effects the isotope ratio was measured for both silver isotopes

26

(107, 109 amu), which roughly reflects the natural abundance (data not shown). Thus, no spillover

27

compensation was necessary.20

28

Calibration and Quantification of Cell-Associated AgNPs by Mass Cytometry. Cells

29

measured in ‘cell mode’ were acquired for 300-900 s. For calibration purposes, different aqueous

30

AgNPs suspensions (0, 0.05, 0.1 and 1 µg Ag L-1) were measured. To calculate the number of

107Ag

signals were


12

Page 13 of 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

AgNPs per cell, the measured median 107Ag intensity obtained from a sample was divided by the

2

median

3

sample/Median

4

For measurements in ‘liquid mode’, digested samples were properly diluted, and their 107Ag signal

5

intensities were acquired for 30 s. Different AgNO3 standard solutions (0, 0.1, 1.0 and 10.0 µg Ag

6

L-1 in 2% HNO3) were used for calibration. The silver concentration for each cell sample (in ng

7

Ag/mL) was determined by comparing the measured of 107Ag intensities to the set of Ag standard

8

solutions. The absolute Ag mass per cell was further calculated as fg Ag/cell = (ng Ag /mL divided

9

by the number of cells/mL) x 1x106. The absolute mass of Ag per cell was further correlated with

10

the absolute Ag mass of a single 50 nm AgNP revealing the absolute number of AgNPs per single

11

cell (AgNPs/cell = fg Ag per cell/0.743 fg per AgNP). To compare quantitative results from

12

digested samples measured in ‘liquid mode’ with single-cell analyses measured in cell mode, we

13

considered the median

14

193Ir+107Ag+,

15

Supporting Information

16

Ag intensities with or without DNA staining, gating strategy, expression of cell surface markers,

17

107Ag/193Ir

18

Instrumental features, AgNPs concentrations/number of events, Excel worksheets quantification

19

results, dead cell frequencies

107Ag

intensity obtained from a single AgNP (AgNPs/cell = Median

107Ag

107Ag

Intensitycell

IntensityAgNPs).

107Ag

signal intensities from AgNPs associated to cells, defined as

and from free AgNPs, 193Ir-107Ag+ (Figure S-4).

–plots of cells, MC signal of AgNPs vs cells

20 21

Acknowledgments. A.L.-S.O. was supported by the Marie Curie Actions, Horizon 2020 (project

22

NanoCytox, 1184). S.B. acknowledges funding by the German Federal Ministry of Education and

23

Research (BMBF) within the framework of the e:Med research and funding concept

24

(sysINFLAME, grant # 01ZX1306B). This work was also supported by the DFG-project “

25

GERMANET” – German Network for Mass Cytometry, grant Me 3644/5-1 and by the Leibniz

26

Science Campus Berlin Chronic Inflammation (www.chronische-entzuendung.org). Furthermore,

27

the project was funded by the German Federal Institute for Risk Assessment (1329-534).


13


Page 14 of 17

1

Authors’ Contributions Statement: A.L.-S.O., A.H., N.J., A.L., A.G., and S.B. designed the study,

2

provided critical feedback and helped shape the research and analysis. D.W. performed cell culture

3

experiments, A.P. performed sample preparation for mass cytometric analysis, A.L.-S.O. and S.B.

4

contributed to sample preparation, CyTOF measurements and data interpretation, S.B. set up the

5

mass cytometer, and A.L.-S.O., S.B., A.H., N.J., and A.G. wrote the manuscript.

6


14

Page 15 of 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2 3

REFERENCES 1.

Vance, M. E.; Kuiken, T.; Vejerano, E. P.; McGinnis, S. P.; Hochella, M. F.; Rejeski, D.;

4

Hull, M. S. Nanotechnology in the real world: Redeveloping the nanomaterial consumer

5

products inventory. Beilstein Journal of Nanotechnology 2015, 6, 1769–1780.

6

http://doi.org/10.3762/bjnano.6.181.

7

2.

8 9

Mirza, A.Z.; Siddiqui, F.A. Nanomedicine and drug delivery: a mini review. Int Nano Lett 2014 4: 94. https://doi.org/10.1007/s40089-014-0094-7.

3.

Ivask, A.; Mitchell, A. J.; Malysheva, A.; Voelcker, N. H.; Lombi, E. Methodologies and

10

approaches for the analysis of cell-nanoparticle interactions. WIREs Nanomed.

11

Nanobiotechnol. 2017. doi:10.1002/wnan.1486

12

4.

Guehrs, E.; Schneider, M.; Günter, C.M.; Hessing, P; Heitz, K.; Wittke, D.; López-Serrano,

13

A.; Jakubowski, N.; Plendl, J.; Eisebitt, S.; Haase, A. Quantification of silver nanoparticle

14

uptake and distribution within individual human macrophages by FIB/SEM slice and view.

15

J. Biotechnology. 2017, 15: 21.

16

5.

17 18

Cerchiaro, G.; Manieri, T. M; Bertuchi, F. R. Analytical methods for copper, zinc and iron quantification in mammalian cells. Metallomics. 2013, 5, 1336-45.

6.

Milic, M.; Leitinger, G.; Pavicic, I.; Avdicevic, M. Z.; Dobrovic, S.; Goessler, W; Vrcek, I.

19

V. Cellular uptake and toxicity effects of silver nanoparticles in mammalian kidney cells. J.

20

Appl. Toxicol. 2014, 35, 581-592.

21

7.

22 23

Mueller, L.; Traub, H.; Jakubowski, N.; Drescher, D.; Baranov, V. I.; Kneipp, J. Trends in single-cell analysis by use of ICP-MS. Anal. Bioanal. Chem. 2014. 406, 6963-6977.

8.

Meyer, S.; Lopez-Serrano, A.; Mitze, H.; Jakubowski, N.; Schwertle, T. Single-cell analysis

24

by ICP-MS/MS as a fast tool for cellular bio-availability studies of arsenite (iAsIII),

25

Metallomics. 2018, 10, 73-76.

26 27

9.

Lopez-Serrano Oliver, A.; Baumgart, S.; Bremser, W.; Flemig, S.; Wittke, D.; Grützkau, A.; Luch, A.; Haase, A.; Jakubowski, N. Quantification of silver nanoparticles up-taken by


15


Page 16 of 17

1

single cells using inductively coupled plasma mass spectrometry in the single cell

2

measurement

3

10.1039/C7JA00395A

mode.

J.

Anal.

At.

Spectrom.,

2018,

33,

1256-1263,

DOI:

4

10. Bandura, R. D.; Baranov, I. V.; Ornatsky, I. O.; Antonov, A.; Kinach, R.; Lou, X.; Pavlov,

5

S.; Vorobiev, S.; Dick, J. E.; Tanner, D. S. Mass Cytometry: Technique for Real Time Single

6

Cell Multitarget Immunoassay Based on Inductively Coupled Plasma Time-of-Flight Mass

7

Spectrometry Anal. Chem. 2009 81 (16), 6813-6822, DOI: 10.1021/ac901049w

8 9

11. Cossarizza et al. Guidelines for the use of flow cytometry and cell sorting in immunological studies. Eur. J. Immunol. 2017, 47: 1584-1797. DOI: 10.1002/eji.201646632

10

12. Yang, S. Y-S.; Atukorale, P. U.; Moynihan, K. D.; Bekdemir, A.; Rakhra, K.; Tang, L.;

11

Stellacci, F.; Irvine, D.J. High-throughput quantitation of inorganic nanoparticle

12

biodistribution at the single-cell level using mass cytometry. Nat. Commun. 2017, 8, 14069.

13

13. Ivask, A.; Mitchell, A. J.; Hope, C. M.; Barry, S.C.; Lombi, E.; Voelcker, N. H. Single cell

14

level quantification of nanoparticle-interactions using mass cytometry. Anal. Chem. 2017,

15

89, 8228–8232.

16

14. Miyashita, S.; Groombridge, A.S.; Fujii S.; Takatsu, K. C.; Inagaki, K. Time-resolved ICP-

17

MS measurement: a new method for elemental and multiparametric analysis of single cells.

18

Anal. Sciences. 2014, 30, 219-224

19

15. Haase, A.; Tentschert, J.; Jungnickel, H.; Graf, P.; Mantion, A.; Draude, F.; Plendl, J.; Goetz,

20

M.E.; Galla, S.; Masi, A.; Huenemann, A. F.; Taubert, A.; Arlinghaus, H.F.; Luch, A.

21

Toxicity of silver nanoparticles in human macrophages: uptake, intracellular distribution and

22

cellular responses. J. Phys. Conf. Ser. 2011, 304, 1.

23

16. Tricot, S.; Meyrand, M.; Sammicheli, C.; Elhmouzi-Younes, J.; Corneau, A.; Bertholet, S.;

24

Malissen, M.; Le Grand, R.; Nuti, S.; Luche, H.; Cosma. A.; Evaluating the Efficiency of

25

Isotope Transmission for Improved Panel Design and a Comparison of the Detection

26

Sensitivities of Mass Cytometer Instruments. Cytometry Part A. 2015. 87 (4), 357-368.

27


16

Page 17 of 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

17. Massimo, A; De Monte, L; Scirea, A; Gruarin, P; Tandon, N. N.; Sitia, R. Synthesis,

2

Processing, and Intracellular Transport of CD36 during monocyte differentiation. J.Biol.

3

Chem. 1996, 271, 3, 1770-1775

4

18. Baumgart, S., Peddinghaus, A., Schulte-Wrede, U., Mei, H. E.; Grützkau, A. OMIP-034:

5

Comprehensive immune phenotyping of human peripheral leukocytes by mass cytometry

6

for monitoring immunomodulatory therapies. Cytometry Part A. 2017, 91, 34–38.

7 8 9 10

19. Leipold, M.D.; Maecker, H.T. Mass cytometry: Protocol for daily tuning and running cell samples on a CyTOF mass cytometer. J Vis Exp. 2012: e4398. 20. Chevrier, Stéphane et al. “Compensation of Signal Spillover in Suspension and Imaging Mass Cytometry” Cell systems vol. 6,5 2018: 612-620.e5


17

Mass Cytometry Enables Absolute and Fast Quantification of Silver

Recommend Documents