Generation of Mo-DCs

From monocyte isolation to DC characterization

Introduction

As professional antigen-presenting cells, dendritic

cells (DCs) are a cornerstone of the adaptive immune

system. Various clinical trials have shown the great

immunotherapeutic potential of DC-based vaccines in the

treatment of tumors.¹

Immature DCs residing in peripheral tissues, including

blood, have the capacity to take up tumor-associated

antigens, for example. Following maturation, DCs

up-regulate their antigen processing machinery, express

a variety of cell surface markers that are involved in the

formation of immunological synapse, and migrate from the

periphery to draining lymph nodes where they present the

antigen to T cells². Both antigen presentation and triggering

of CD28 on T cells by the DC costimulatory markers, CD80

and CD86³, are required to induce an antigen-specific

response. Upon stimulation T cells up-regulate the

expression of CD40 ligand (CD40L) and proliferate to

counteract the disease that is associated with the particular

pathogenic antigen. Binding of CD40L on activated T helper

h) cells to CD40 on mature DCs provides a feedback signal

that leads to the secretion of IL-12 by DCs, which in turn

drives T cell polarization towards the T

h1 lineage and the

antitumor response mediated by cytotoxic T cells.

Large numbers of DCs for basic research and

immunotherapies can be generated in vitro from

monocytes

5,6

. Differentiation/maturation protocols have

been described by various groups

7–10

. To obtain functional

monocyte-derived DCs (Mo-DCs) that possess the distinct

phenotype and function of natural DCs, it is crucial to

follow reliable procedures using high-quality reagents.

This application note describes all the steps for the

generation of Mo-DCs and their phenotypic and functional

characterization.

Material and methods

Isolation of monocytes

CD14

monocytes were isolated from peripheral blood

mononuclear cells (PBMCs) from healthy donors by

MACS® Technology using CD14 MicroBeads (Miltenyi Biotec).

Purity and recovery were determined by labeling cells with

CD14-FITC antibodies and subsequent flow cytometry

analysis on a MACSQuant® Analyzer 10 (Miltenyi Biotec).

Cell viability was determined according to propidium

iodide (PI) staining.

Differentiation and maturation of monocyte-derived

dendritic cells (Mo-DCs)

The generation of Mo-DCs can be achieved by using various

protocol formats, manually and automatically, at various

scales. Here we describe the procedure for 6-well plates as

an example. On day 0, isolated monocytes (3×10 cells) are

cultured in 3 mL of complete medium (RPMI 1640, 2 mM

L-glutamine, 1% autologous plasma), supplemented

7,8

with

250 IU/mL IL-4 and 800 IU/mL GM-CSF, and incubated at

37°C and 5% CO. On day 2, a volume of 1.5 mL is removed

from the culture and centrifuged. Cells are resuspended in

1.5 mL of complete medium supplemented with the 2-fold

concentration of IL-4 and GM-CSF and put back into the

original culture. On day 6, a volume of 1.5 mL is removed

from the culture and centrifuged. Cells are resuspended in

1.5 mL medium supplemented

9,10

with 2000 IU/mL IL-6, 400

IU/mL IL-1β, 2000 IU/mL TNF-α, and 2 µg/mL PGE, put back

into the original culture, and cultured for another 24 hours.

On day 7, viability, yield, and absolute cell count of mature

Mo-DCs (mMo-DCs) is determined by flow cytometry via

light scatter signals and PI fluorescence. For the gating

strategy see figure 1.

For all experiments shown in this application note, Mo-DCs

were generated using cytokines from Miltenyi Biotec.

Analysis of cell morphology

Monocytes as well as immature Mo-DCs (imMo-DCs) and

mMo-DCs were placed in a 6-well plate and allowed to

sediment. Images of the cells were captured using a light

microscope with phase-contrast at a 400× magnification.

Immunophenotyping of monocytes and Mo-DCs

by flow cytometry

To analyze cell surface expression of various maturation

markers, co-stimulatory molecules, and receptors for

chemokines and antigens, the monocytes, imMo-DCs,

and mMo-DCs were labeled with specific monoclonal

antibodies (table 1). Non-specific antibody binding was

assessed using appropriate isotype controls. The mean

fluorescence intensity (MFI) of the respective markers was

determined by flow cytometry on a MACSQuant Analyzer

10 using the MACSQuantify™ Software (Miltenyi Biotec).

Cell debris and dead cells were excluded from the analysis

based on scatter signals and PI fluorescence. All antibodies

and Propidium Iodide Solution were from Miltenyi Biotec.

Cell surface antigen Clone Fluorochrome

CD1a HI149 PE

CD14 TÜK4 FITC

CD25 3G10 APC

CD HB APC

CD REA PE

CD REA PE

CD REA APC

CD FM PE

CD DCN APC

CD (CCR) REA APC

CD (DC-SIGN) REA APC

HLA-DR AC FITC

HLA-ABC REA FITC

Table : Antibodies used for monocyte and Mo-DC

immunophenotyping. All antibodies were from Miltenyi Biotec.

Antigen uptake capacity

To assess the pinocytosis capacity, 1×10 monocytes,

imMo-DCs, or mMo-DCs were incubated with FITC-labeled

dextran (1 mg/mL) in complete medium (RPMI, 2 mM

L-glutamine, 1% autologous plasma) for 5, 10, 20, 30, and

60 minutes at 37 °C. To check for non-specific binding of

FITC-dextran to the cell surface, a control sample was kept

on ice for 60 min. After 60 min, all samples were washed

twice (centrifugation at 300×g, 5 min, 4 °C) with ice-cold

PBS supplemented with 1% fetal calf serum (FCS) and finally

suspended in PBS supplemented with 0.5% BSA. Samples

were kept on ice until flow cytometry analysis. Uptake of

the FITC-dextran was determined by measuring the mean

fluorescence intensity (MFI) of FITC by flow cytometry.

Dead cells were excluded from the analysis

by PI fluorescence. Specific uptake of FITC-dextran was

calculated by subtracting the MFI of the control sample

that was incubated on ice from the MFI of the samples

incubated at 37 °C.

Migratory capacity of mMo-DCs

CCR7-dependent migration of mMo-DCs towards CCL19 was

tested in 24-well Transwell® Plates (Corning; pore size 5 µm).

The mMo-DCs were resuspended in RPMI 1640 with 10%

FCS at a density of 5×10 cells/mL, and 200 µL were placed

in the upper compartment of a Transwell Plate. The lower

compartment was filled with 600 µL of complete medium

supplemented with Human CCL19 (MIP-3β) (Miltenyi Biotec)

at different concentrations. After 3 h the cells contained in

the lower compartment were harvested and counted on a

MACSQuant® Analyzer 10.

Isolation of human naive CD4

T cells for mixed

lymphocyte reaction (MLR)

Naive allogeneic CD4

T cells were isolated from PBMCs

using the Naive CD4

T Cell Isolation Kit II, human

(Miltenyi Biotec) according to the instructions provided in

the data sheet. Purity of the isolated cells was determined

by labeling with i) CD4-PE or ii) CD45RO-APC and CD45RA-

PE antibodies (all from Miltenyi Biotec) and subsequent

analysis by flow cytometry using the MACSQuant

Analyzer10.

Assessment of the T cell priming capacity

of Mo-DCs in MLR

The capacity of Mo-DCs to induce proliferation of naive

T cells was measured in MLR. To this end, 5×10 purified

naive CD4

T cells were suspended in 400 µL PBS, and

labeled with 100 µL of a 10 µM CellTrace™ Violet solution

(Life Technologies®) for 5 min at RT. Subsequently, the cells

were washed once with 1 mL FCS and three times with

2 mL MLR medium (RPMI 1640, 2 mM L-glutamine, non-

essential amino acids, 0.1 mM sodium pyruvate, 5% human

AB serum). Finally, the CD4

T cells were suspended in MLR

medium at a density of 5×10 cells/mL.

Monocytes, imMo-DCs, and mMo-DCs were suspended

in MLR medium at a density of 1×10 cells/mL and

serially diluted according to table 2. The different cell

dilutions (100µL per well) were placed in a 96-well plate.

Subsequently, the labeled T cells (100 µL per well) were

added and cultured for 7 days at 37 °C, 5% CO.

Proliferation of CD4

T cells was determined by measuring

the fluorescence of CellTrace Violet by flow cytometry.

Cell debris and dead cells were excluded from the analysis

by scatter signals and PI fluorescence.

Mo-DC/monocyte

density (cells/mL)

Number of Mo-DCs/

monocytes per well

Ratio of Mo-DCs/

monocytes to

Tcells

2.5×10 25,000 1:2

1.25×10 12,500 1:4

6.25×10 6,250 1:8

.×10 , :

.× , :

.×³  :

Table : Serial dilution of Mo-DCs/monocytes for MLR assay.

The number of naive T cells was constant at ,.

Quantitation of IL-12 and IL-10 secreted

by mMo-DCs after stimulation with CD40L

The mMo-DCs were suspended in medium (RPMI, 2 mM

L-glutamine, 1% autologous plasma) at a density of 1×10

cells/mL, and 5×10 cells were used for the assay performed

in 96-well plates.

To measure the secretion of IL-12p70 and IL-10, mMo-DCs

were stimulated with soluble Human CD40-Ligand (16µg/

mL; Miltenyi Biotec) or in a co-culture with J558L cells

expressing CD40L (7×10, 14×10, or 28×10 cells/well) for

comparison. Mo-DCs were stimulated for 24 h at 37 °C, 5%

CO. Subsequently, the cell supernatants were collected

and centrifuged (300×g; 10 min) to remove any cells. The

concentrations of IL-12p70 and IL-10 were determined by

specific ELISAs (eBioscience). The CD40L-expressing J558L

cell line was kindly provided by Professor Marina Cella

(Washington University, St. Louis, MO, USA)

Results

Isolation of monocytes to high purities

CD14

monocytes were isolated from PBMCs by MACS

Technology to a purity of 96.8±3.8%. The proportion

of CD14

cells that were recovered from the PBMC

fraction amounted to 81.3±8.8%%, and the viability

was 97.5±1.4% (fig. 1).

Percentage

100

Viability PurityRecovery

97.5 +/– 1.4%

81.3 +/– 8.8%

96.8 +/– 3.8%

Figure : Isolation of CD

monocytes. Purity, recovery (i.e.,

percentage of CD

cells in the purified fraction in relation to the

original fraction), and viability of enriched CD

cells are shown.

Cells were labeled with a CD-FITC antibody and analyzed by

flow cytometry.

Differentiation of monocytes into mature Mo-DCs

Purified monocytes were used to generate Mo-DCs

according to the protocol for 6-well plates. On day 7, cells

were analyzed by flow cytometry using the gating strategy

depicted in figure 2. The viability of mMo-DCs, evaluated

by excluding PI

dead cells, amounted to 95.4±2.4%.

The recovery of mMo-DCs was 23%, calculated based on

the number of originally seeded monocytes (fig. 2).

Side scatter

Forward scatter

97.7%

Forward scatter

Figure : Generation of Mo-DCs. Viability, yield (i.e., mMo-DC count

in relation to initial monocyte count), and total count of mMo-DCs

generated by using the protocol for -well plates (n= ). Data were

obtained by flow cytometry on the MACSQuant Analyzer  following

the gating strategy shown: First cells were gated according to their

scatter properties. Subsequently, dead cells were excluded from the

analysis by PI fluorescence.

Mo-DCs assume the characteristic DC morphology

during differentiation

Dendritic cells have a distinct morphology characterized

by many cellular processes. To evaluate whether monocytes

assume this morphology during differentiation and

maturation to Mo-DCs, we analyzed the cells on days

0, 6, and 7 of cell culture, both by microscopy and flow

cytometry. On day 0, the cells had a spherical shape,

which is normal for monocytes. After 6 days the cells

showed cytoplasmic protrusions, which were even

more pronounced after maturation on day 7 (fig. 3A).

Moreover, size and granularity of the cells increased

during differentiation and maturation, which is reflected

in increased forward and side scatter signals in the flow

Mo-DC viability and yield (%)

Absolute cell count

100 1.5×10

1.0×10

5.0×10

0 0

Mo-DC

viability

Absolute

Mo-DC count

Mo-DC

yield

cytometry analysis (fig. 3B).

Monocytes

(day 0)

Immature Mo-DCs

(day 6)

Mature Mo-DCs

(day 7)

Forward scatter

Side scatter

Figure : Morphology of monocytes, imMo-DCs, and mMo-DCs.

Cells were analyzed on days , , and  of differentiation/maturation.

(A) Images were captured on a light microscope using phase contrast

at a × magnification. (B) Assessment of relative cell size and

granularity by flow cytometry using forward and side scatter channels,

respectively. , events are shown for each dot plot.

Mature Mo-DCs express characteristic

DC surface markers

Flow cytometry analysis of cell surface markers showed

that in vitro generated Mo-DCs assumed the typical DC

phenotype (fig. 4). During differentiation monocytes

down-regulated the expression of CD14. In addition,

mMo-DCs expressed various DC markers that are involved

in the formation of immunological synapse between DC

and naive T cells, including the costimulatory proteins

CD80 and CD86, the cell adhesion molecule CD54, and

antigen-presenting molecules MHC I (HLA-ABC) and MHC II

(HLA-DR). Mature Mo-DCs also expressed the DC activation

markers, CD83, CD25, and CD40, which were up-regulated

accordingly. CCR7, which is required for migration of DCs

to draining lymph nodes, was also up-regulated. The

highest expression levels of the antigen uptake receptors,

CD209 and CD206, were detected on imMo-DCs (fig. 5),

corresponding to their antigen uptake function.

Fluorescence intensity

Relative cell number

MonocytesimMo-DCsmMo-DCs

HLA-ABCHLA-DR

CD209CD14

100

250

200

150

100

800

600

400

200

150

100

MFI

Fluorescence intensity

MonocytesimMo-DCsmMo-DCs

CD83CD86CD80CD40

100

15 150

10 100

5 50

0 0

400

300

200

100

Relative cell number

MFI

Fluorescence intensity

MonocytesimMo-DCsmMo-DCs

CCR7CD1aCD25CD54

100

15 150

10 100

5 50

0 0

400

300

200

100

Relative cell numberMFI

Monocytes imMo-DCs mMo-DCs

Figure : Immunophenotyping of monocytes, imMo-DCs, and

mMo-DCs.

Cells were labeled on days , , and  with antibodies specific

for the respective markers and analyzed by flow cytometry. The histograms

show the results from one representative experiment Markerspecic

and isotype control labeling are shown in red and black respectively

The plots below the histograms depict the MFI for each marker on

monocytes imMODCs and mMoDCs in six independent experiments

Immature Mo-DCs have the highest antigen

uptake capacity

Antigen uptake by antigen-presenting cells can occur

specifically via receptor-mediated endocytosis or non-

specifically via pinocytosis or phagocytosis. To assess

the pinocytosis capacity of in vitro generated Mo-DCs in

comparison to monocytes, cells were cultured at 37 °C for

up to 1 h in the presence of FITC-dextran. Non-specific

binding of FITC-dextran to the cell surface was determined

by incubating samples on ice throughout the procedure.

The MFI of FITC was analyzed by flow cytometry after

various time points (fig. 5A). Subtraction of MFI

Ice

(samples

on ice) from MFI (samples at 37 °C) allowed for assessment

of the relative antigen uptake capacities (fig. 5B). Immature

Mo-DCs had the highest antigen uptake capacity, reflected

in a continuous increase in MFI over the entire time course.

Mature Mo-DCs from day 7 showed decreased antigen

uptake activity compared to imMo-DCs throughout the

time course. The antigen uptake was further decreased

when Mo-DCs were matured for additional 2 days (day 9).

Monocytes showed a negligible antigen uptake capacity

compared to Mo-DCs.

Additionally, flow cytometry analysis showed that

imMo-DCs exhibited the highest expression of CD206 and

CD209 (fig. 5C). These results correlate with the finding that

imMo-DCs lose their antigen uptake function upon maturation.

FITC-dextran

Rel. cell number

37 °C Ice

0 min 5 min 10 min 20 min 30 min 60 min 60 min

MFI – MFI

Ice

Monocytes

imMo-DCs

mMo-DCs (day 7)

mMo-DCs (day 9)

105 20 30 60

Time (min)

MFI

150

100

Monocytes mMo-DCs

(day 9)

mMo-DCs

(day 7)

imMo-DCs

CD206

CD209

Figure : Antigen uptake capacity of monocytes and Mo-DCs.

(A) Flow cytometry results from a representative experiment with

imMo-DCs cultured at  °C in the presence (red) or absence (black)

of FITC-dextran for the indicated amounts of time. Non-specific binding

was determined by incubating cells on ice. (B) Relative FITC-dextran

uptake was calculated by subtracting the MFI of cells incubated for

 minutes on ice (MFI

Ice

) from the MFI of cells incubated at  °C (MFI).

Results are shown for monocytes, imMo-DCs, and mMo-DCs (days 

and ). (C) Monocytes and Mo-DCs were labeled with CD-APC or

CD-APC antibodies and analyzed by flow cytometry.

Mature Mo-DCs migrate towards CCL19

Upon activation of DCs in the periphery, the cells migrate

to the draining lymph nodes where they encounter naive

T cells. Migration depends on the expression of CCR7

on the DC surface

. The CCR7 ligand CCL19 acts as a

chemoattractant for DCs and is expressed in the lymph

node areas characterized by high T cell densities. Figure 4

indicates that CCR7 expression was up-regulated during

Mo-DC maturation. To measure the migration capacity of

in vitro generated mMo-DCs, we used Transwell Plates.

Figure 6 shows that mMo-DCs migrated towards a CCL19

stimulus in a dose-dependent manner.

Number of cells that migrated

CCL19 concentration (ng/mL)

60,000

40,000

20,000

200 100 50 25

12.5 6.25 3.125 1.56 0.78 0.39

Figure : Migration of mMo-DCs towards a CCL stimulus. The

mMo-DCs (× cells) were placed in the upper compartment of a

-well Transwell Plate. Media containing different concentrations

of CCL were placed in the lower compartment. After  h the total

number of cells that migrated to the lower compartment was

determined by flow cytometry.

Mature Mo-DCs have the capacity to prime naive T cells

After migration to the lymph nodes, DCs can induce the

proliferation of naive T cells. We tested the capacity of

monocytes and Mo-DCs to induce T cell proliferation in

an MLR. To this end, we isolated allogeneic naive

CD45RA

CD45RO

–

T cells to high purities by MACS®

Technology (fig. 7A) and labeled their plasma membrane

with CellTrace Violet (fig. 7B). After coculturing the labeled

T cells with monocytes or Mo-DCs for 7 d, we determined

the cell numbers of T cells and analyzed the CellTrace Violet

staining by flow cytometry. As with each division of the

Tcells the dye gets more diluted in the plasma membrane,

an increase in unlabeled cells indicates a high T cell

proliferation rate. In contrast to monocytes both imMo-DCs

and mMo-DCs increased the number of CellTrace Violet–

negative T cells in the culture (fig. 7C). This correlated with

higher T cell numbers, as determined by cell counting.

Tcell proliferation was highest when mMo-DCs were used

as antigen-presenting cells (fig. 7D), which is in line with

the aforementioned result (fig. 4A) showing that receptors

involved in T cell priming are up-regulated on Mo-DCs

upon maturation.

Rel. cell number

CellTrace Violet

CD45RA

CD45RO

Enriched naive T cells

CD4

96%

A B

CellTrace Violet

Rel. cell number

Monocytes imMo-DCs mMo-DCs

Number of T cells

3×10

2×10

1×10

Monocytes

imMo-DCs

mMo-DCs

20100 4030 6050 70

T cell: Mo-DC ratio

Figure : Induction of T cell proliferation by Mo-DCs. (A) Naive

CD

T Cells were isolated from PBMCs by MACS Technology. Isolated

cells were stained with CD-PE or CDRA-PE/CDRO-APC antibodies

and analyzed by flow cytometry using the MACSQuant Analyzer

. (B) Isolated naive T cells were labeled with CellTrace Violet and

analyzed by flow cytometry. (C) CellTrace Violet–labeled naive T cells

were cocultured with monocytes, imMo-DCs, and mMo-DCs at a ratio

of : for  d and analyzed by flow cytometry. (D) Naive T cells were

cocultured with imMo-DCs and mMo-DCs at various ratios. After d

the numbers of T cells were determined by flow cytometry based

on scatter signals. Dead cells were excluded from the analysis by PI

fluorescence. One of three representative experiments is shown.

Mature Mo-DCs secrete IL-12 upon stimulation

with soluble CD40L

In general, DCs have the capacity to secrete both

pro-inflammatory and immunoregulatory cytokines,

depending on the stimulus received. T cell–mediated

CD40L stimulation of Mo-DCs induces the production

of T

h1-polarizing IL-12, but also the secretion of the

immunosuppressive IL-10

12-14

. However, for studies towards

the development of cancer therapies it is desirable to

generate Mo-DC populations secreting high amounts of

IL-12 and low amounts of IL-10 as these cells generate a more

effective antitumor response via induction of T

h1 cells.

To determine the capacity of in vitro generated Mo-DCs

to secrete IL-12 and IL-10, we stimulated mMo-DCs under

various conditions for one day: i) incubation with soluble

Human CD40-Ligand (Miltenyi Biotec), which forms

multimers in vitro, and ii) coculture with a J558L cell line

expressing CD40L. Stimulation with soluble CD40L led to

production of high levels of IL-12 and low levels of IL-10.

Co-culture with CD40L-expressing J588L cells resulted in an

overall increase in secretion of both IL-12 and IL-10 (fig. 8A).

However, the ratio of IL12/IL-10 was higher after stimulation

with soluble CD40L than after coculture with the cell line

(fig. 8B). Thus, recombinant human CD40L multimers

represent an attractive alternative to CD40L-transfected

cell lines, allowing for CD40 stimulation of Mo-DCs under

defined conditions.

Cytokine concentration (pg/mL)

800

700

500

300

100

600

400

200

Control J558

CD40L***

Soluble

CD40L

J558

CD40L*

J558

CD40L**

IL-12p70

IL-10

Ratio IL-12p70:IL-10

Soluble

CD40L

J558

CD40L***

J558

CD40L**

J558

CD40L*

Figure : IL- and IL- secretion by mMo-DCs upon stimulation

with CDL. (A) The mMo-DCs were cultured for  h in the absence

or presence of soluble CDL ( µg/mL) or a J cell line expressing

CDL (*× cells/well, **× cells/well, ***× cells/well).

IL-p and IL- concentrations in the culture supernatant were

determined by ELISA. (B) The ratios of IL-p vs. IL- were calculated

based on the results shown in (A).

Conclusion

• This applications note describes procedures that cover

a complete workflow for the generation and phenotypic

and functional analysis of mMo-DCs.

• CD14 MicroBeads enable the isolation of viable CD14

monocytes to high purities, with high yields. The

isolated monocytes can be easily differentiated into

Mo-DCs.

• A straightforward and reliable differentiation/

maturation protocol based on cytokines from Miltenyi

Biotec enables the effective generation of mMo-DCs.

• In vitro generated imMo-DCs and mMo-DCs possess the

characteristics of DCs in terms of i) morphology, ii)

surface marker expression, iii) antigen uptake capacity,

iv) migration towards a CCL19 stimulus, v) induction of

Tcell proliferation, and vi) capacity to secrete IL-12 in

response to CD40L stimulation.

• Comprehensive phenotypic analysis is accomplished

using flow cytometry tools from Miltenyi Biotec,

including the powerful MACSQuant Analyzer 10 and

a wide range of MACS Antibodies.

• Recombinant human CCL19 from Miltenyi Biotec is a

potent chemoattractant for CCR7-expressing Mo-DCs.

• CD40L from Miltenyi Biotec effectively stimulates

Mo-DCs to secrete IL-12. The resulting ratio of IL-12:IL-10

is at least as high as with a CD40L-expressing cell line.

Product Order no.

Cell isolation

CD14 MicroBeads, human 130-050-201

CD MicroBeads, human – lyophilized --

Naive CD

T cell Isolation Kit II, human --

Flow cytometry

MACSQuant Analyzer  --

MACSQuantify Software --

Anti-HLA-DR-FITC, human *

Anti-HLA-ABC-FITC, human *

CDa-PE, human *

CD-PE, human *

CD-FITC, human *

CD-APC, human *

CD-APC, human *

CDRA-PE, human *

CDRO-APC, human *

CD-PE, human *

CD-PE, human *

CD-APC, human *

CD-PE, human *

CD (CCR)-APC, human *

CD-APC, human *

CD (DC-SIGN)-APC, human *

Propidium Iodide Solution --

Cell culture

Human CCL (MIP-β), research grade --**

Human CD-Ligand, premium grade --**

Human GM-CSF, premium grade --**

Human IL-β, premium grade --**

Human IL-, premium grade --**

Human IL-, premium grade --**

Human TNF-α, premium grade --**

* for different package sizes and additional conjugates, visit

www.miltenyibiotec.com/antibodies

** Order numbers are provided for  µg sizes.

For different quality grades and additional package sizes, visit

www.miltenyibiotec.com/cytokines

References

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