immuno-monitoring using the scepter™ 2.0 cell counter and software pro

8/6/2019 Immuno-monitoring using the Scepter 2.0 Cell Counter and Software Pro

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Immuno-monitoring usingthe Scepter 2.0 Cell Counterand Software Pro

Application Note

AbstractBiological samples such as primary isolates or cultured

cells are oten heterogeneous mixtures o cells that

dier by type and/or unction. Such dierences in

cellular attributes are most commonly determined by

multicolor uorescent antibody detection o cell type-

specifc surace marker(s) using ow cytometry. Notably,

in addition to variations in protein expression, many

cell types and physiological states are also uniquely

distinguishable on the basis o size alone. The ability to

identiy population subsets on the basis o phenotypic

dierences and urther determine their relative

requencies (and concentrations) is critical to many

aspects o research.

The Scepter cell counter combines the ease o automated

instrumentation and the accuracy o the Coulter

principle o impedance-based particle detection in an

aordable, handheld ormat. The instrument uses a

combination o analog and digital hardware or sensing,

signal processing, data storage, and graphical display. The

precision-made, consumable polymer sensor has a laser-

drilled aperture in its cell sensing zone that enables the

EMD Millipore is a division of Merck KGaA, Darmstadt, Germany.

Amedeo Cappione, Ph.D.1; Emily Crossley2; Nagaraja Thirumalapura, DVM, Ph.D.3; and Debra Hoover, Ph.D.11EMD Millipore; 2Dept. o Pathology, Dept. Microbiology & Immunology, University o Texas Medical Branch;3Dept. o Pathology, University o Texas Medical Branch

instrument to use the Coulter principle to discriminate

cell diameter and volume at submicron and subpicoliter

resolution, respectively.

Using a sensor with a 40 m aperture, the Scepter cell

counter can accurately and precisely count a broad range

o cell types, including small cells (> 3 m in diameter)

and peripheral blood mononuclear cells (PBMC)1. This

article outlines three examples o experiments using

the Scepter cell counters sensitive size-discriminating

capability to demonstrate rapid, qualitative assessment

o individual cell population requencies in complex cell

mixtures.
http://www.millipore.com/life_sciences/flx/scepter&open&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/life_sciences/flx/scepter&open&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/life_sciences/flx/scepter&open&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/life_sciences/flx/scepter&open&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/life_sciences/flx/scepter&open&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/life_sciences/flx/scepter&open&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/life_sciences/flx/scepter&open&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/?cid=BIOS-S-EPDF-1043-1106-DS


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ExamplE 1:Lymphocyte vs. monocyte subset

discrimination in reshly isolated PBMCs

IntroductionThe human immune system is comprised o cell

subsets, with distinct unctional profles, that fght

pathogens. The assessment o immune profles o the

various immune cell subsets, such as lymphocytes andmonocytes, can help identiy molecular signatures

that may acilitate research, ranging rom translational

medicine, prognostic advancements, and early decision-

making tools in vaccine development. The Scepter

cell counter, when used in combination with Scepter

Sotware Pro, provides a tool or rapid determination o

lymphocyte and monocyte concentrations as well as the

relative requency o these cell types in PBMC isolates.

Materials and Methods

Human blood sample prepHuman PBMCs were isolated rom whole heparinized

blood o healthy donors by Ficoll-Paque density

centriugation (GE Healthcare). Briey, 9 mL o blood

was diluted to 25 mL with phosphate-buered saline

(1X EmbryoMax PBS, EMD Millipore). and layered over

15 mL o Ficoll. Samples were centriuged at 400 x g or

30 minutes with no brake, and the resulting PBMC layer

was recovered. The PBMC ractions were washed twice

by centriugation using PBS. Ater fnal spin, cell pellets

were resuspended in PBS or sufcient conductivity

during Scepter cell counting.

Scepter cell counting

The Scepter cell counter was used to count samples

ollowing the simple on-screen instructions or each step

o the counting process. Briey, the user attaches a

40 m sensor, depresses the plunger, submerges the

sensor into the sample, then releases the plunger

drawing 50 L o cell suspension through the cell-

sensing aperture. The Scepter cell counter detects each

cell passing through the sensors aperture, calculates cell

concentration, and displays a histogram as a unction o

cell diameter or volume on its screen.

Scepter 2.0 sotware was then used to upload test fles

rom the device and perorm subsequent data analysis

to determine the concentrations and relative cellrequencies or the lymphocyte and monocyte ractions.

Guava easyCyte cell counting

10 L o each PBMC sample was diluted in 190 L PBS.

Samples were then analyzed on a guava easyCyte HT

system to determine the concentrations and relative cell

requencies or the lymphocyte and monocyte ractions.

Cell viability determination using

guava ViaCount assay

10 L o each PBMC sample was mixed with 190 L

ViaCount assay reagent, incubated or 5 minutes at room

temperature (RT). Viability data were acquired on a guava

easyCyte instrument using guava ExpressPro sotware.

Cell surface staining and subset determination

For each sample, 100,000 PBMCs were resuspended

in 100 L PBS+0.1% bovine serum albumin (BSA).

To distinguish the discrete cell subsets present in

PBMC samples, they were stained with the ollowing

combination o uorescently labeled antibodies: CD3-PE

(T cells), CD19-Alexa Fluor 488 (B cells), CD16/CD56-

APC (NK cells), and CD14-PECy7 (monocytes) (antibodies

all rom eBioscience). Singly stained samples and isotype

controls were included with each staining set to ensure

proper instrument setup. Samples were incubated

at RT or 20 minutes, washed twice with PBS, then

resuspended in 200 L PBS prior to acquisition. Samples

were analyzed (3000 cells/sample well) on a guava

easyCyte HT system using guava ExpressPro sotware.

01
http://www.millipore.com/search.do?q=embryomax&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/search.do?q=embryomax&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/search.do?q=embryomax&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/search.do?q=embryomax&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/search.do?q=embryomax&cid=BIOS-S-EPDF-1043-1106-DS


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ResultsViability assessment of PBMC

Freshly prepared or rozen PBMC samples consisted

o live cells, dead cells, and a considerable amount

o cellular debris. The debris component varied with

respect to properties inherent to the blood sample as

well as the methods o storage and PBMC preparation.

To understand the relative proportions o live cells, deadcells and debris, reshly isolated PBMC were stained with

ViaCount reagent.

ViaCount reagent distinguished viable and non-viable

cells based on dierential permeabilities o two DNA-

binding dyes. The cell-permeant nuclear dye stained

all nucleated cells, while the cell-impermeant viability

dye brightly stained dying cells. Cellular debris was not

stained by either dye.

Results rom a representative assay are presented

in Figure 1A. In this example, more than 95% o the

cells were viable. The three components were also

distinguishable on the basis o particle size. The

histograms in Figure 1B show the distribution o particles

in the dierent ractions as unction o orward scatter,

a ow cytometry-based correlate o particle size. While

there was some overlap between size distributions o

dead cells and debris, the live cells constituted a distinct

raction made up o two dierently-sized cell types.

Quantifying subsets of PBMCs

(T cells, B cells, NK cells, monocytes)

PBMC can be subdivided into many distinct cell types

based on the varying expression o specifc surace

markers and unctional capabilities (or example, cytokine

secretion). The abundance and relative distribution o

these subsets are unctions o developmental state as

well as overall health. Two main populations o PBMC

are lymphocytes and monocytes. The lymphocyte subset

can be urther subdivided into T cells (CD3+), B cells

(CD19+), and NK cells (CD16/56+). Monocytes can be

distinguished rom all lymphocytes on the basis o CD14

expression.

Figure 1. Representative ow

cytometry data demonstrating

size-based discrimination o live

vs dead cells. (A) live cells (blue),

dead cells (red), and debris (green)

ractions were defned using

ViaCount reagent. (B) The our

histograms show the relativedistribution o each raction as a

unction o particle size (orward

scatter).

Figure 2. Representative data

rom ow cytometry analysis o

multicolor staining perormed on

PBMC ractions. (A) Live cells (red)

are distinguished rom debris/

dead cells (green) on the basis o

orward vs. side scatter. (B) Live

cells rom (A) are ractionated in

the 2 plots based on variations in

expression o CD14, CD3, CD16/56,

and CD19. (C) The our histograms

show the localization o each

cell raction with regards to the

two peaks defned by orward

scatter. As shown, the three major

lymphocyte subsets are ound

predominantly in the smaller-

cell peak (blue) while the CD14+

monocytes are restricted to the

large cell raction (pink).

10e4

10e3

10e2

Red

Fluorescence

10e1

10e0

10e0 10e1

Yellow Fluorescence

10e2 10e3

M1

M2

M3

10e4

M3

M1 M1

M1 M1

100

otal EventsA B Live Cells

Dead Cells Debris

75

50

25

0

0 2500 5000

Forward Scatter

Count

7 50 0 1 00 00

100

75

50

25

0

0 2500 5000

Forward Scatter

Count

7 50 0 1 00 00

100

75

50

25

0

0 2500 5000

Forward Scatter

Count

7 50 0 1 00 00

50

38

25

13

0

0 2500 5000

Forward Scatter

Count

7 50 0 1 00 00

Live Cells

Debris/

RBCM1

10000

7500

5000

Side

Scatter

2500

0

0 2500

Forward Scatter

5000 7500 10000

M1 Monocytes

T cellsM2

M1

M2

NK cells

B cells

A B C

CD3+10e4

10e3

10e2

10e1

10e0

75

100

50

25

0

0

1 0e 0 1 0e 1 1 0e 2 1 0e 3 1 0e 4

2500 5000

Forward Scatter

Count

CD3-PE (T-cells)

CD14-PECy7(Monos)

10e4

10e3

10e2

10e1

10e0

1 0e 0 1 0e 1 1 0e 2 1 0e 3 1 0e 4

CD19-A488 (B-cells)

CD56-APC(NKCells)

7500 10000

M1

M2

M3

CD14+

75

100

50

25

0

0 25 00 5 00 0

Forward Scatter

Count

7500 10000

M1

M2

M3

M1

M2

M3

CD19+

75

100

50

25

0

0 2 50 0 5 000

Forward Scatter

Count

7500 10000

D16/56+

75

100

50

25

0

0 25 00 5 00 0

Forward Scatter

Count

7500 10000

M1

M2

M3

M1

M2

M3


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To investigate the distribution o these our subsets with

regards to size, PBMCs were stained with uorescent

antibodies specifc or each surace marker (Figure 2). The

dot plots confrmed defnition o each subset by staining

with each cell-type specifc marker. Figure 2C indicated

that B, T, and NK cells were generally smaller in size (low

orward scatter) while CD14+ monocytes were larger in

size (higher orward scatter).

To examine the ability o the 40 m Scepter sensor

to discriminate lymphocytes rom monocytes, PBMC

ractions were isolated rom resh human blood by

Ficoll gradient centriugation. Diluted samples were

analyzed using both a guava easyCyte ow cytometer

and the Scepter cell counter. Representative histogram

plots are presented in Figure 3. In each case, three main

peaks were distinguishable by each analysis method,

with greater peak resolution in the ow cytometry

data, particularly in the separation o lymphocytes rom

debris. The peaks correspond to lymphocytes (small cells),

monocytes (large cells), and a debris/dead cell raction.

Across the nine PBMC samples analyzed (Table 1),

the average mean cell diameters were 7.230.30 m

and 10.020.20 m or lymphocytes and monocytes,

respectively. Resulting values were consistent with

previously reported size ranges2

. In addition, cellrequencies were determined by three methods:

Scepter diameter plot, guava easyCyte orward

scatter, and antibody staining. The Scepter values

slightly underestimated the lymphocyte raction while

overestimating the monocyte subset. This is likely the

result o the Scepter cell counters comparatively lower

resolution as well as subjectivity and user bias in the

placement o gates defning each subset. Overall, there

was good agreement between the dierent analytical

techniques with values varying by


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ExAMPE 2:

Human T-cell activation

IntroductionIn vivo, T lymphocytes are activated and induced

to prolierate upon binding o the T cell receptor to

antigen-presenting cells. In response to stimulation,

T cells undergo physical, biological, and phenotypicchanges, including increased cell size, secretion o

cytokines, and up-regulation o CD25 surace expression,

ultimately culminating in the production o specifcally-

tuned immune responses. Elevated CD25 expression

levels are a late-stage indicator o T-cell activation4.

Assays measuring changes in T- and B-cell activation

are commonly used to identiy patterns o immune

response in clinical diagnostics as well as therapeutic

development.

The in vivo immune response by T cells can be mimickedex vivo by binding T cells to co-immobilized anti-CD28

and anti-CD3 monoclonal antibodies3. PBMCs can

also be activated through exposure to more generic

inducers o cell prolieration, such as the plant lectin

phytohemagglutinin (PHA) or pokeweed mitogen. Ex

vivo activation enables detailed studies o the molecular

mechanisms regulating T-cell activation and response.

Materials and MethodsCell culture/activation

Human PBMC ractions were isolated as previously

described. Prior to culture, initial cell concentrations were

determined using the Scepter cell counter. All culturing

experiments were perormed in RPMI 1640 supplemented

with 10% etal bovine serum (FBS) (R10) in the absence

o antibiotics. PBMC (400,000 cells/mL, 24-well plates)

were stimulated or two days in the presence o soluble

anti-CD28 mAb (clone 28.2, BD Pharmingen, 2 g/mL)

on plates pre-coated with anti-CD3 mAb (clone HIT3a;

BD Pharmingen, 10 g/mL). Mitogenic stimulation was

carried out in the presence o 2 g/mL PHA (Sigma).

Unstimulated control cultures were also analyzed.

Scepter cell countingSample acquisition and data analysis was perormed

as previously described. Briey, ollowing stimulation, a

small aliquot o each culture was harvested, diluted in

PBS, then analyzed on the Scepter cell counter. Test fles

were uploaded and analyzed using Scepter Sotware Pro

to determine the degree o cell activation (% activated

cells) as well as concentrations or both unstimulated

and activated ractions.

CD25 staining for activation

Following two-day stimulation, cultures were harvested,

washed twice with PBS, and then counted using the

Scepter cell counter. For each sample, 100,000 cells were

resuspended in 100 L PBS+0.1% BSA. To distinguish

the activated T-cell raction, samples were stained with

anti-CD3-PE (T cells) and anti-CD25-APCeFluor780

(antibodies rom eBioscience). Singly stained samples and

Isotype controls were included with each staining set to

ensure proper instrument setup. Samples were incubated

at RT or 20 minutes, washed twice with PBS, then

resuspended in 200 L PBS prior to acquisition. Samples

were analyzed (3000 cells/sample well) on a guava

easyCyte HT system using guava ExpressPro sotware.

02


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The dot plots in Figure 4B showed three main

populations o cells: resting T cell (CD3+/CD25-),

activated T cells (CD3+/CD25+), and non-T cells (CD3-/

CD25-). By comparison, cultures stimulated with CD3/

CD28 Ab demonstrated signifcantly greater levels o

T-cell activation than those exposed to PHA. Control

cultures showed very low requencies o activated cells.

Under all conditions, CD25 expression also correlatedwith an increase in overall cell size (Figure 4C).

ResultsTo address the potential use o the Scepter cell counter

or rapid qualitative monitoring o immune cell

activation as a unction o the cell size shit, reshly

isolated PBMCs were stimulated in culture using two

dierent mechanisms: (1) CD3/CD28 antibody-mediated

co-stimulation o the T cell receptor (TCR) and (2)

mitogenic stimulation by PHA.

Figure 4. Expression o the cell activation marker CD25 correlates with an increase in cell size. (A) An elliptical gate was used to identiy live cells. (B) Dot plots depict the ractionation o live

cells on the basis o CD3 and CD25 expression. (C) The orward scatter histogram plots demonstrate that expression o CD25 is restricted to the larger-cell raction or all culture conditions.

CD25 eFluor780

CD3-PE

Side

Scatter

Side

Scatter

Forward Scatter

Side

Scatter

Forward Scatter

CD3-PE

CD3-PE

CD25 eFluor780

CD25 eFluor780

Forward Scatter

Side

Scatter

Forward Scatter

Side

Scatter

Forward Scatter

Side

Scatter

Forward Scatter

Side

Scatter

Forward Scatter

Side

Scatter

Forward Scatter

Side

Scatter

Forward Scatter

A B C


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the live, unstimulated raction. This overlap was clearly

demonstrated in the untreated sample, in which two

peaks were seen in the ow cytometry histogram plot o

total cells (Marker R5). By comparison, the live cell-only

histogram showed a single peak within R5. The resulting

data clearly demonstrated the subset o activated T cells

but could not be used discriminate the live, unstimulated

T cell population.

In Figure 5, the Scepter cell counters ability to detect cell

activation was compared to that o the guava easyCyte

ow cytometry platorm. While the Scepter cell counter

was able to detect the presence o the larger, activated

cell raction in both stimulated cultures, the device

was unable to simultaneously discriminate the smaller

unstimulated population in these samples. This was most

likely due to the presence o a large number o deadcells (red cells in Figure 5A) that overlapped in size with

Figure 5. Representative data demonstrating size-based discrimination o resting vs. activated cells in control and stimulated PBMC cultures. (A) Live cell (blue), dead cell (red), and de bris

(green) ractions were defned using ViaCount reagent. (B) The two histograms in each row show the relative distribution o each raction as a unction o particle size (orward scatter). Plots

are based on total events and live cells, respectively. (C) Histogram data or each sample ollowing analysis using the Scepter cell counter with 40 m sensor.

Side

Scatter

Side

Scatter

Forward Scatter

Side

Scatter

Forward Scatter

Forward Scatter

Count

Forward Scatter

Count

Forward Scatter

Count

Diameter (m)

Count

Diameter (m)

Count

Diameter (m)

Count

Forward Scatter

Count

Forward Scatter

Count

Forward Scatter

Count

Forward Scatter

A B C


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8

ExAMPE 3:Splenic cell shift in murine

model of Ehrlichiosis

IntroductionHuman monocytotropic ehrlichiosis (HME) is an

emerging tick-borne disease caused by the obligately

intracellular pathogen Ehrlichia chaffeensisthat resides

in mononuclear phagocytes. HME initially maniestsas nonspecifc u-like symptoms but can progress to

lie-threatening toxic shock-like syndrome with anemia,

thrombocytopenia, and multi-organ ailure. The presence

o extensive inammation in the absence o bacterial

burden suggests that mortality is the consequence o

unregulated immunopathology5,6. In this study, a murine

model o HME based on inection o C57BL/6 mice

with Ehrlichia muriswas used to investigate aspects

o the immune response, in particular, changes to the

population dynamics o splenocytes.

Figure 6. Cell surace staining defnes cell subsets within the mouse

spleen. Forward vs. side scatter plots reveal the presence o two main

splenocyte populations. (A) The raction containing larger cells is

comprised primarily o leukocytes (B, NK, and T cells), while the raction

o small cells (B) is predominantly er ythrocytes (Ter119+).

Materials and methodsInfection of mice with Ehrlichia muris

Six- to eight-week-old C57BL/6 mice were inected

with Ehrlichia muris (~1 x 104 bacterial genomes) by

the intraperitoneal route. Mice were sacrifced on day

30 post-inection and spleens were harvested. Single-

cell suspensions o splenocytes were prepared using

the GentleMACS Tissue Dissociator ollowing themanuacturers instructions (Miltenyi Biotec Inc., CA).

Determination of splenocyte cell distribution using

the Scepter cell counter

Cells were diluted to appropriate concentrations

(~1 2 x 105 cells/mL) and loaded into the Scepter cell

counter ftted with the 40 M sensor. The gates were

set to exclude red blood cells using a splenocyte sample

rom an uninected, nave mouse. Splenocytes rom three

uninected mice and three mice inected with Ehrlichia

muriswere used. Data were analyzed using Scepter

Sotware Pro.

Determination of splenocyte cell distribution

by ow cytometry

Splenocytes were incubated with a uorescent viability

dye (Near IR LIVE/DEAD Fixable Dead Cell Stain Kit,

Invitrogen, CA) or 10 minutes in PBS at RT and protected

rom light. Cells were washed and blocked with Fc Block

(BD Biosciences, CA) and purifed rabbit, rat and mouse

IgGs (Jackson ImmunoResearch, PA) in ow cytometry

buer (PBS + 2% FBS + 0.09% sodium azide). Cells were

then incubated with uorescently labeled antibodies

to the surace markers B220 (B cells) and Ter119

(Erythrocytes) (BD Biosciences, CA and eBioscience, CA,

respectively) and analyzed by ow cytometry.

ResultsSingle-cell suspensions o mouse splenocytes were

analyzed by multicolor ow cytometry (Figure 6). Size-

based discrimination using orward scatter confrmed the

presence o two distinct cell populations in all samples.Staining with antibodies specifc or B-cells (B220) and

erythrocytes (Ter-119) demonstrated that erythrocytes

were restricted to the subset containing smaller cells,

while the raction o large cells was composed primarily

o lymphocytes.

03

A

B

SSC

FSC

SSC

FSC

B200-

PerCP-

Cy5.5

Ter119-APC

B200-

PerCP-

Cy5.5

Ter119-APC

B cells

T/NK cells

1

2

70

27

Erythroid

194

32


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Flow cytometry-derived histograms o total cells showed

the presence o three distinct populations corresponding

to debris, lymphocytes (large cells) and erythrocytes

(small cells). Comparative analysis o splenic cell isolates

rom Ehrlichia-inected and control mice revealed an

overall shit in the relative cell distribution. Specifcally,

inected samples showed an increase in the percentage

o erythrocytes when compared to the lymphocyte

subset (Figure 7B and Table 2). In contrast, the Scepter

histogram was able to detect only two peaks; the third

peak corresponding to the debris raction had particle

sizes below the lower limit o detection. In addition, the

smaller erythrocytes (red blood cells) were at the limits

o detection or the Scepter cell counter and were not

Table 2. Total lymphocyte and erythrocyte subset requencies rom six individual splenocyte isolates (three control, three inected). Aliquots rom each

sample were analyzed using a ow cytometer and Scepter cell counter.

Count

Dead/debris

Erythroid

Lymphocyte

Count

Diameter (m)

Count

A B C

SSC

FSC FSC

FSC

SSC

FSC

Diameter (m)

Figure 7. Size-based ractionation o mouse splenocytes. Untreated andEhrlichia-inected samples were analyzed using a ow cytometer (A, B) and

using the Scepter cell counter (C). Three distinct peaks corresponding to lymphocytes, ery throcytes, and debris can be visualized using ow cytometry.

For the Scepter cell counter, the particle size o the debris peak is below the level o detection or the 40 m sensor.

Scepter Flow Cytometry

Test Sample Erythrocyte ymphocyte Erythrocyte ymphocyte

Control-1 27 73 16 84

Control-2 29 71 21 79

Control-3 22 78 14 86

Inected - 1 40 60 42 58

Inected - 2 30 70 29 71

Inected - 3 33 67 38 62


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10

counted, skewing the erythrocyte counts. However, the

Scepter cell counters quantitation o splenocyte counts

and percentages were in very good agreement with

the values determined by ow cytometry. An overall

assessment o cell concentration values indicated the

shit was due to an expansion o the erythrocyte subset

in the spleen while lymphocyte values remained constant(Table 3). These fndings were consistent with previously

published results7.

Overall ConclusionsEvaluation o immune responsiveness to viral, bacterial,

cellular and environmental exposure is an important

tool in assessment o pathogenicity and toxicity .

The availability o simplifed methods or identiying

activation status o cells and or measuring dierences

in the relative requencies (and concentrations) o more

than one cell type in samples or co-cultures is very

useul or accelerating research in these felds.

We have presented data indicating that both the guava

easyCyte benchtop ow cytometry platorm as well as

the Scepter cell counter can determine such dierences

in resh primary isolates and cultured samples. While

the ow cytometry system is the more sophisticated

and quantitative tool, the Scepter is a rapid tool that can

be used at the hood to provide very good insight into

cellular responses in concert with existing methods.

The unctionality o Scepter counting is a result o the

precision-engineered sensor and the unique handheld

instrumentation based upon the Coulter principle. Theability o the Scepter device to ensure reproducible cell

counts improves data quality during experimental setup

and downstream cell-based analyses.

Table 3. Total lymphocyte and erythrocyte cell concentration or all six samples.

References

1. Smith, J et al. The New Scepter 2.0 Cell Counter Enables the

Analysis o a Wider Range o Cell Sizes and Types With High

Precision. EMD Millipore Cellutions 2011 Vol. 1: p 19-22.

2. Daniels, V. G., Wheater, P. R., & Burkitt, H. G. (1979).

Functional Histology: A Text and Colour Atlas. Edinburgh:

Churchill Livingstone. ISBN 0-443-01657-7.3. Prager, E. et al. (2001) Induction o Hyporesponsiveness and

Impaired T Lymphocyte Activation by the CD31 Receptor:

Ligand Pathway in T-Cells. J. Immunol. 166: 2364-2371.

4. Levine, B. L., et al. (1997). Eects o CD28 Costimulation on

Long-term Prolieration o CD4+ T-cells in the Absence o

Exogenous Feeder Cells. J. Immunol. 159:5921 5930.

5. Olano, J. P., G. Wen, H. M. Feng, J. W. McBride, and D. H.

Walker. 2004. Histologic, Serologic, and Molecular Analysis

o Persistent Ehrlichiosis in a Murine Model. Am. J. Pathol.

165:9971006.

6. Paddock, C. D., and J. E. Childs. 2003. Ehrlichia chaeensis:

a Prototypical Emerging Pathogen. Clin. Microbiol. Rev.

16:3764.

7. MacNamara, K.C., Racine, R. Chatterjee, M., Borjesson, D, and

Winslow, G.M. (2009) Diminished Hematopoietic Activity

Associated with Alterations in Innate and Adaptive Immunity

in a Mouse Model o Human Monocytic Ehrlichiosis. Inect.

And Immunity. 77:4061-69.

Cell Concentration (105)

Test Sample Erythrocyte ymphocyte

Control-1 1.86 4.65

Control-2 2.61 5.54

Control-3 2.17 5.78

Inected 1 4.3 5.22

Inected 2 2.82 4.93

Inected - 3 3.5 5.84


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Description Quantity Catalog No.

Scepter 2.0 Handheld Automated Cell Counter

with 40 m Scepter Sensors (50 Pack) 1 PHCC20040

with 60 m Scepter Sensors (50 Pack) 1 PHCC20060

Includes:

Scepter Cell Counter 1

Downloadable Scepter Sotware 1

O-Rings 2

Scepter Test Beads 1 PHCCBEADS

Scepter USB Cable 1 PHCCCABLE

Scepter Sensors, 60 m 50 PHCC60050

500 PHCC60500

Scepter Sensors, 40 m 50 PHCC40050

500 PHCC40500

Universal Power Adapter 1 PHCCP0WER

Scepter O-Ring Kit, includes 2 O-rings and 1 flter cover 1 PHCC0CLIP

Description Catalog No.

Guava ViaCount Reagent 4000-0041

FlowCellect Human Lymphocyte ZAP-70 Characterization Kit FCIM025122

FlowCellect Mouse FoxP3 Treg Identifcation Kit FCIM025126

guava Cell Toxicity Reagents Kit 4500-0230

FlowCellect Human FOXP3 Treg Characterization Kit FCIM025118

FlowCellect Mouse Th1 Intracellular Cytokine Kit FCIM025123



FlowCellect Mouse Th1/Th2 Intracellular Cytokine Kit FCIM025137

FlowCellect Mouse Th1/TH17 Intracellular Cytokine Kit FCIM025138

Ordering Information

Scepter 2.0 Cell Counter

Description Quantity Catalog No.

High-Throughput Sampling Instruments

guava easyCyte 8HT Base System 1 0500-4008

guava easyCyte 6HT/2L Base System 1 0500-4007

guava easyCyte 5HT Base System 1 0500-4005

PCA-96 Base System 1 0100-8710

Single Sampling Instruments

easyCyte 5 Base System 1 0500-5005

easyCyte 6-2L Base System 1 0500-5007

easyCyte 8 Base System 1 0500-5008

Guava easyCyte Benchtop Flow Cytometers

FlowCellect Flow Cytometry Assay Kits and Milli-MarkFluorescent Conjugated Antibodies for Immunology

Discover the power o ow cytometry or multiparametric analysis o the immune system:

visit www.millipore.com/CellularAnalysisor complete listings o our ully validated conjugates

and assay kits optimized or guava easyCyte systems.
http://www.millipore.com/catalogue/item/PHCC20040&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/PHCC20060&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/PHCCBEADS&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/PHCCCABLE&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/PHCC60050&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/PHCC60500&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/PHCC40050&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/PHCC40500&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/PHCCP0WER&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/PHCC0CLIP&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/4000-0041&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/FCIM025122&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/FCIM025126&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/4500-0230&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/FCIM025118&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/FCIM025123&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/FCIM025124&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/FCIM025125&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/FCIM025137&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/FCIM025138&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/0500-4008&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/0500-4007&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/0500-4005&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/0100-8710&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/0500-5005&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/0500-5007&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/0500-5008&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/cellbiology/flx1/insightful_cell_analysis&open&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/cellbiology/flx1/insightful_cell_analysis&open&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/FCIM025138&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/FCIM025137&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/FCIM025125&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/FCIM025124&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/FCIM025123&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/FCIM025118&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/4500-0230&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/FCIM025126&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/FCIM025122&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/4000-0041&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/cellbiology/flx1/insightful_cell_analysis&open&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/0500-5008&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/0500-5007&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/0500-5005&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/0100-8710&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/0500-4005&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/0500-4007&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/0500-4008&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/PHCC0CLIP&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/PHCCP0WER&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/PHCC40500&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/PHCC40050&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/PHCC60500&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/PHCC60050&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/PHCCCABLE&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/PHCCBEADS&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/PHCC20060&cid=BIOS-S-EPDF-1043-1106-DShttp://www.millipore.com/catalogue/item/PHCC20040&cid=BIOS-S-EPDF-1043-1106-DS


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EMD Millipore and the M logo are trademarks o Merck KGaA, Darmstadt, Germany. Guava, ViaCount andEmbryoMax are registered trademarks and Scepter, FlowCellect, guava easyCyte, and Milli-Mark are trademarkso Millipore Corporation. GentleMACS is a trademark o Miltenyi Biotec, Inc.Alexa Fluor is a registered trademark o Lie Technologies, Inc. Fc Block is a trademark o BD Biosciences.Lit No. AN3322EN00 BS-GEN-11-04565 Printed in the USA. 05/2011

2011 Millipore Corporation. All rights reserved.

www.millipore.com/CellularAnalysis

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In the U.S. and Canada, call toll-ree 1-800-645-5476

For other countries across Europe and the world,

please visit www.millipore.com/oices

For Technical Service, please visit

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immuno-monitoring using the scepter™ 2.0 cell counter and software pro

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