Macrophage cell culture€¦ · Macrophage cell culture Fig. 1: PromoCell macrophage cell culture portfolio. Overview of possible applications with PromoCell's macrophage media and

Macrophage cell culture

Fig. 1: PromoCell macrophage cell culture portfolio. Overview of possible applications with PromoCell's macrophage media and cryopreserved macrophages.Abbreviations: IC = immune complexes, IFN = interferon, IgG = immunoglobulin G, GC = glucocorticoids, (G)M-CSF = (granulocyte/)macrophage colony stimulating factor, IL = interleukin, LPS = lipopolysaccharide, PBMC = peripheral blood mononuclear cells, TAM = tumor-associated macrophages (freshly isolated from tumor tissue), TGF = transforming growth factor.

Generation of macrophages

10d

or

24h

M2 macrophagesM(GC)

M(TGFβ)M(IL-10)

M(IC+LPS)M(IL-4)

M1 macrophagesM(IFN-γ)

M(LPS+IFN-γ)M(LPS)

Activation factor(s)+

Freshly isolated monocytes

Isolation of tumor-associated macrophages

Malignant (human)tumor tissue

TAM

Macrophage Base Medium DXF

PromoCell GmbHHeidelberg, Germanywww.promocell.com

Addition by user:SupplementMix:

Date: __________

Cat. No.: C-28057Lot No.: Sample Volume 250 mlExp.: Sample Store at: + 4°C

For research use only for research use only for research use only for research use only


M2-Macrophage Generation

Medium DXFM2-Macrophage GenerationMedium DXF (Ready-to-use)



Date: __________



or+ 2% human AB serum

M2 macrophagesM(-)-M-CSF

Polarized macrophagesUnpolarized macrophages

Polarized, activated macrophages (optional)

M1 macrophagesM(-)-GM-CSF

CryopreservedM1 or M2 macrophages

M0 / Mϕ macrophages




Date: __________




M1- or M2-Macrophage Generation Medium DXF

M2-Macrophage GenerationMedium DXF (Ready-to-use)



Date: __________






Date: __________



M1- or M2-Macrophage Generation Medium DXF




Date: __________






Date: __________



PromoCell provides a full range macrophage cell culture portfolio including media systems to generate macrophages from fresh peripher-al blood mononuclear cells (PBMCs) or to isolate tumor-associated macrophages (TAMs) from tumor tissues, assay-ready cryopreserved macrophages as well as subsequent macrophage analysis tools.

Macrophage cell culture Macrophage cell culture 32

In VItro Macrophage generatIon

Ready-to-use: M1/M2-Macrophage

Generation Media DXF

The PromoCell M1-Macrophage Gen-eration Medium DXF contains GM-CSF and allows for the generation of M1-polarized macrophages, whereas the M2-Macrophage Generation Medi-um DXF contains M-CSF and produces M2-polarized macrophages. Both media are ready-to-use and tested in-house for successful differentiation of M1-/M2-macrophages via flow cytometry.

User-customizable:


The Macrophage Base Medium DXF is the user-customizable version of the Macrophage Generation Media product line. It comes without cytokines as a universally applicable MDM culture sys-tem featuring a fully user-customizable macrophage differentiation and activa-tion process (see Fig. 3 and Tab. 1 for suggestions).

Defined and xeno-free formulation

All PromoCell Macrophage Generation Media DXF are defined and xeno-free media formulations, free of all ani-mal-derived components and substanc-es of human origin with human serum albumin as the only exception.

Efficient isolation of monocytes:

Monocyte Attachment Medium

The Monocyte Attachment Medium allows for the efficient adherence se-lection of monocytes from freshly iso-lated human mononuclear cells while maintaining optimal cell health. Thus, the time-consuming and costly immu-nomagnetic purification of monocytes is not necessary.

Product Size Catalog Number

Monocyte Attachment Medium 250 ml C-28051



Macrophage Generation Medium DXF




M1-Macrophage Generation Medium DXF (incl. GM-CSF) 250 ml C-28055

M2-Macrophage Generation Medium DXF (incl. M-CSF) 250 ml C-28056

Macrophage Base Medium DXF 250 ml C-28057

GM-CSF 10 µg C-60422

M-CSF 10 µg C-60420

M1/M2 Ready-to-use Macrophage Generation Media


Fig. 2: Non-activated human mono-cyte-derived macrophages (MDM) dif-ferentiated directly from PBMC. Mono-cytes were purified using the Monocyte Attachment Medium and differentiated in Macrophage Base Medium DXF contain-ing 100 ng/ml GM-CSF for 10 days.

Intended monocyte-derived macrophages (MDM) activation state by addition of the respective activation factor(s)*

Polarization state M2M1

IFN-γ LPS+IFN-γ LPS — — — DEX IL-10 IgG+LPS IL-4 —TGF-β1 Activation factor(s)

GM-CSF or M-CSF GM-CSF or M-CSF GM-CSF or M-CSF GM-CSF 2% hAB serum M-CSF M-CSF M-CSF M-CSF M-CSF 2% hAB serumM-CSF Differentiation factor

M(IFN-γ) M(LPS+IFN-γ) M(LPS) M(-)-GM-CSF M0 / Mϕ M(-)-M-CSF M(GC) M(IL-10) M(IC+LPS) M(IL-4) TAMM(TGFβ) MDM activation stateProduct

Macrophage Base Medium DXF (C-28057)

M1-Macrophage Generation Medium DXF incl. GM-CSF (C-28055)

M2-Macrophage Generation Medium DXF incl. M-CSF (C-28056)

Tab. 1: In vitro macrophage generation with the PromoCell macrophage media system. Overview of the wide spectrum of macrophage polarization and activation states achievable with PromoCell's macrophage generation media. Abbreviations: DEX = dexamethasone, IC = immune complexes, IFN = interferon, IgG = immunoglobulin G, GC = glucocorticoids, (G)M-CSF = (granulocyte/)macrophage colony stimulating factor, h = human, IL = interleukin, LPS = lipopolysaccharide, PBMC = peripheral blood mononuclear cells, TAM = tumor-associated macrophages (freshly isolated from tumor tissue), TGF = transforming growth factor. *Nomenclature by used activation factor(s)

In Vitro Macrophage Generation– How to choose the right medium for your macrophages –

Fresh PBMC

Unpolarized

Polarized, non-activated

Polarized, activated

Adherent monocytes


M2 macrophagesM(GC)

M(TGFβ)M(IL-10)

M(IC+LPS)M(IL-4)

M2 macrophagesM(GC)

M(TGFβ)M(IL-10)

M(IC+LPS)M(IL-4)




M(LPS+IFN-γ)M(LPS)


M(LPS+IFN-γ)M(LPS)





Date: __________




Pola

riza

tion

Act

ivat

ion

(opt

iona

l)

+ M-CSF

+ G

M-C

SF

Unpolarized macrophagesM0/Mϕ

+ 2%

hum

an

AB se

rum

Customized




Date: __________







Date: __________






Date: __________



M2-Macrophage Generation Medium DXF

(incl. M-CSF)


(incl. GM-CSF)

M(GC) M(IL-10) M(IC+LPS) M(IL-4)M(TGFβ)

Abbreviations: DEX = dexamethasone, IC = immune complexes, IFN = interferon, IgG = immunoglobulin G, GC = glucocorticoids, (G)M-CSF = (granulocyte/)macrophage colony stimulating factor, h = human, IL = interleukin, LPS = lipopolysaccharide, PBMC = peripheral blood mononuclear cells, TAM = tumor-associated macrophages (freshly isolated from tumor tissue), TGF = transforming growth factor. *Nomenclature by used activation factor(s)

+ L

PS

+ L

PS +

IFN

-γ

+ IF

N-γ

+ D

EX

+ T

GFβ

+ Ig

G +

LPS

+ IL

-10

+ IL

-4


+ D

EX

+ T

GFβ

+ Ig

G +

LPS

+ IL

-10

+ IL

-4

M(IFN-γ) M(LPS+IFN-γ) M(LPS)

+ L

PS

+ L

PS +

IFN

-γ

+ IF

N-γ


www.promocell.com | PromoCell GmbH | Sickingenstr. 63/65 | 69126 Heidelberg | GermanyPhone: +49 6221-649 34 0 | Fax: +49 6221-649 34 40 | [email protected]

More information and detailed protocols are available in the application note: Convenient large-scale differentiation of fully customized monocyte-derived macrophages from human PBMC in vitro

GM-CSF

M-CSF

—

Intended monocyte-derived macrophages (MDM) activation state by addition of the respective activation factor(s)*Cytokines

in medium






M1-Macrophage Generation Medium DXF (C-28055)


TAM

Customized




Date: __________




+ 2

% h

uman

AB

seru

m

Malignant (human) tumor tissue

In Vitro Macrophage Generation– How to choose the right medium for your macrophages –

Fresh PBMC

Unpolarized

Polarized, non-activated

Polarized, activated

Adherent monocytes


M2 macrophagesM(GC)

M(TGFβ)M(IL-10)

M(IC+LPS)M(IL-4)

M2 macrophagesM(GC)

M(TGFβ)M(IL-10)

M(IC+LPS)M(IL-4)




M(LPS+IFN-γ)M(LPS)


M(LPS+IFN-γ)M(LPS)





Date: __________




Pola

riza

tion

Act

ivat

ion

(opt

iona

l)

+ M-CSF

+ G

M-C

SF

Unpolarized macrophagesM0/Mϕ

+ 2%

hum

an

AB se

rum

Customized




Date: __________







Date: __________






Date: __________




(incl. M-CSF)


(incl. GM-CSF)


Abbreviations: DEX = dexamethasone, IC = immune complexes, IFN = interferon, IgG = immunoglobulin G, GC = glucocorticoids, (G)M-CSF = (granulocyte/)macrophage colony stimulating factor, h = human, IL = interleukin, LPS = lipopolysaccharide, PBMC = peripheral blood mononuclear cells, TAM = tumor-associated macrophages (freshly isolated from tumor tissue), TGF = transforming growth factor. *Nomenclature by used activation factor(s)

+ L

PS

+ L

PS +

IFN

-γ

+ IF

N-γ

+ D

EX

+ T

GFβ

+ Ig

G +

LPS

+ IL

-10

+ IL

-4


+ D

EX

+ T

GFβ

+ Ig

G +

LPS

+ IL

-10

+ IL

-4


+ L

PS

+ L

PS +

IFN

-γ

+ IF

N-γ


www.promocell.com | PromoCell GmbH | Sickingenstr. 63/65 | 69126 Heidelberg | GermanyPhone: +49 6221-649 34 0 | Fax: +49 6221-649 34 40 | [email protected]

More information and detailed protocols are available in the application note: Convenient large-scale differentiation of fully customized monocyte-derived macrophages from human PBMC in vitro

GM-CSF

M-CSF

—

Intended monocyte-derived macrophages (MDM) activation state by addition of the respective activation factor(s)*Cytokines

in medium








TAM

Customized




Date: __________




+ 2

% h

uman

AB

seru

m

Malignant (human) tumor tissue

+

+ +

SupplementMix

SupplementMix

GM-CSF or

M-CSF

GM-CSF or

M-CSF

availableseparately

+

Fig. 3: In vitro macrophage generation with the PromoCell macrophage media system. Overview of the wide spectrum of macrophage polarization and activation possibilities with our media system. Abbreviations: DEX = dexamethasone, IC = immune complexes, IFN = interferon, IgG = immunoglobulin G, GC = glucocorticoids, (G)M-CSF = (granulocyte/)macrophage colony stimulating factor, h = human, IL = interleukin, LPS = lipopolysaccharide, PBMC = peripheral blood mononuclear cells, TGF = transforming growth factor.

( )

Macrophage cell culture4

assay-ready: cryopreserVed Macrophages

Macrophage Culture Made Easy:

Cryopreserved Macrophages

No time-consuming in vitro generation Ready-to-use after 24h Convenient access to individual donors Optional activation possible Long-term culture is supported with full functionality

Being part of PromoCell's unique range of macrophage culture products, cryo-preserved human macrophages are now available as a reliable source of stan-dardized cells in a ready-to-use format allowing for full experimental flexibility (Fig. 4). The frozen macrophages are produced in PromoCell´s well-proven M1/M2-Macrophage Generation Media DXF and are available as fully quali-fied M1-(hMDM-GMCSF(-)) or M2- (hMDM-MCSF(-)) polarized cells. The cells can be seeded directly into fi-bronectin-coated multiwell-plates, dishes and flasks. After plating, the mac-rophages can be maintained as biolog-ically functional, adherent cultures (see Fig. 4) for several weeks. Optionally, user-customizable activation of the cells can be performed.

Each lot is tested for cell morphology, adherence rate and viability. Further-more, they are characterized by flow cytometric analysis of relevant markers (Fig. 5): M1: CD68+, CD 80+

M2: CD68+, CD163+

Coat with Fibronectin(C-43060)

Optional: macrophage activation (24h after plating)

4h 4h

Culture of cryopreserved human macrophages




Date: __________






Date: __________




(incl. M-CSF; C-28056)


(incl. GM-CSF; C-28055)

Cryopreserved hMDM-GMCSF(-)(C-12914, C-12916)

Cryopreserved hMDM-MCSF(-)(C-12915, C-12917)

M2 macrophagesM(GC)

M(TGFβ)M(IL-10)

M(IC+LPS)M(IL-4)

M2 macrophages


M(LPS+IFN-γ)M(LPS)

M1 macrophages


+ L

PS

+ L

PS +

IFN

-γ

+ IF

N-γ

+ D

EX

+ T

GFβ

+ Ig

G +

LPS

+ IL

-10

+ IL

-4


Activation factor+Activation factor+

Start assay directly(24h after plating)

Start assay after activation

Fig. 4: Culture of PromoCell's cryopreserved human macrophages. The macrophages can be directly used for experiments after thawing and plating. Optional: activation of polarized M1-/M2 macrophages 24h before assay. For more information see our application notes at www.promocell.com/scientific-resources/application-notes. Abbreviations: DEX = dexamethasone, IC = immune complexes, IFN = interferon, IgG = immunoglobulin G, GC = glucocorticoids, (G)M-CSF = (granulocyte/)macrophage colony stimulating factor, h = human, IL = interleukin, LPS = lipopolysaccharide, PBMC = peripheral blood mononuclear cells, TGF = transforming growth factor.


Human M1 Macrophages (GM-CSF), Monocyte-derived, single donor 1.5 Mio C-12914

Human M1 Macrophages (GM-CSF), Monocyte-derived, single donor 5 Mio C-12916

Human M2 Macrophages (M-CSF), Monocyte-derived, single donor 1.5 Mio C-12915

Human M2 Macrophages (M-CSF), Monocyte-derived, single donor 5 Mio C-12917

Fibronectin Solution, human (1 mg/ml) 5 ml C-43060

Cryopreserved Human Macrophages

Custom solutions:

Do you require a custom produced lot or specific donor characteristics? Contact us!

[email protected]

Macrophage cell culture 5

Fig. 7: Human monocyte-de-rived macrophages: transfected with a galactosidase-expressing plasmid.

Fig. 6: Macrophage phagocytosis assay. Day 10 M2-Macrophages were exposed to flourescently labeled E.coli for 24 hours before re-moving non-ingested bacteria. Left: phase contrast image. Right: fluorescence image of the phagocytizing macrophages.




Macrophage Detachment Solution DXF 250 ml C-41330

Phagocytosis Assay Kit (Green E. coli) 100 assays PK-CA577-K963

Phagocytosis Assay Kit (Red E. coli) 100 assays PK-CA577-K964

Phagocytosis Assay Kit (Green Zymosan) 100 assays PK-CA577-K397

Phagocytosis Assay Kit (Red Zymosan) 100 assays PK-CA577-K398

PromoFectin-Macrophage 100 µl PK-CT-2000-MAC-10

PromoFectin-Macrophage 500 µl PK-CT-2000-MAC-50

Macrophage Detachment Solution

The Macrophage Detachment Solution DXF is a non-enzymatic reagent especially optimized for the gentle detachment of strongly adherent macrophages.

Phagocytosis Assay

Our Phagocytosis Assay Kits utilize pre-labeled E. coli or Zymosan particles as a tool for rapid and accurate detection and quantifi-cation of in vitro phagocytosis by fluorescent microscope, spectrophotometer or flow cytometry. Our kits provide a robust screening system for activators and/or inhibitors of phagocytosis and Toll-like Receptor (TLR) ligands.

PromoFectin-Macrophage

PromoFectin-Macrophage has been developed for optimal transfection of the hard-to-transfect macrophages and macrophage-de-rived cell lines such as RAW 264.7 by binding to mannose-specific membrane receptors. It also works with dendritic cells.

Fig. 5: Flow cytometric analysis of PromoCell cryopreserved Human M1 and M2 Macrophages. M1 macro-phages exhibit a CD68+ (99.74%) and CD80+ (83.33%) marker expression profile, typical for M1 macrophages (upper row). M2 macrophages exhibit a CD68+ (99.72%) and CD163+ (94.91%) marker expression profile, typical for M2 macrophages (lower row).

CD68 – CD68 +

positive: 99.74%

100 101 102 103 1040

20

40

60

80

100

CD80 – CD80 +

positive: 83.33%

100 101 102 103 1040

20

40

60

80

100

positive: 99.72%

CD68 – CD68 +

100 101 102 103 1040

20

40

60

80

100 positive: 94.91%

CD163 – CD163 +

100 101 102 103 1040

20

40

60

80

100

M1

M2

Assays and Reagents for Macrophage Cell Culture

PromoCell GmbH

Sickingenstr. 63/6569126 HeidelbergGermany

Email: [email protected]

USA/CanadaPhone: 1 – 866 – 251 – 2860 (toll free)Fax: 1 – 866 – 827 – 9219 (toll free)

DeutschlandTelefon: 0800 – 776 66 23 (gebührenfrei)Fax: 0800 – 100 83 06 (gebührenfrei)

FranceTéléphone: 0800 – 90 93 32 (ligne verte)Téléfax: 0800 – 90 27 36 (ligne verte)

United KingdomPhone: 0800 – 96 03 33 (toll free)Fax: 0800 – 169 85 54 (toll free)

Other CountriesPhone: +49 6221 – 649 34 0Fax: +49 6221 – 649 34 40

© PromoCell GmbH

Cytokines & Activation Factors Size Catalog Number

ELISAs Size Catalog Number

Dexamethasone 1 g PK-CA577-1042-1G

IFN-γ (E.coli) / IFN-γ (Plant) 100 µg* C-60724 / C-60724A

M-CSF (E.coli) / M-CSF (Plant) 10 μg* C-60442 / C-60442A

GM-CSF (E.coli) / GM-CSF (Plant) 20 µg* / 10 μg* C-60424 / C-60420A

Cryo-SFM 30 ml C-29910

IL-1 beta (E.coli) / IL-1 beta (HEK) 10 µg* / 10 μg* C-61120 / C-61121

IL-4 (E.coli) / IL-4 (Plant) / IL-4 (HEK) 20 µg* / 10 µg* / 10 µg* C-61421 / C-61420A / C-61452

IL-6 (E.coli) / IL-6 (CHO) / IL-6 (HEK) 20 µg* / 10 µg* / 10 µg* C-61625 / C-61640 / C-61642

IL-10 (E.coli) / IL-10 (HEK) 10 µg* C-62012 / C-62014

IL-12 (CHO) / IL-12 (HEK) 10 µg* C-62213 / C-62212

IL-13 (E.coli) 10 µg* C-62312

MCP-1 (CCL2) 10 µg* C-64030

MIP-1 alpha (CCL3) / MIP-3 alpha (CCL20) 20 µg* / 20 µg* C-65833 / C-65866

TGF-β1 (E.coli) / TGF-β1 (CHO) / TGF-β1 (HEK) 10 µg* C-63500 / C-63503 / C-63499

*bulk sizes (100/250/500 and 1000 µg) are available on requestMore cytokines/chemokines/growth factors are available at www.promocell.com/cytokines-growth-factors.

TNF-α ELISA Kit, human 96 tests PK-EL-63709

TGF-β1 ELISA Kit, human 96 tests PK-EL-63506

TGF-β2 ELISA Kit, human 96 tests PK-EL-63508

IL-1α ELISA Kit, human 96 tests PK-EL-61106

IL-1 beta ELISA Kit, human 96 tests PK-EL-61128

IL-6 ELISA Kit, human 96 tests PK-EL-61606



IL-12 (p40) ELISA Kit, human 96 tests PK-EL-62215

IL-12 (p40+p70) ELISA Kit, human 96 tests PK-EL-62207

IL-12 (p70) ELISA Kit, human 96 tests PK-EL-62216



IP-10 (CXCL10) ELISA Kit, human 96 tests PK-EL-65521

More ELISAs are available at www.promocell.com/elisas.

Related Products

Cell-based Assays Size Catalog Number

Cell Migration/Chemotaxis Assay Kit (8 µm, 24-well) 12 Assays PK-CA577-K909






More products and information are available at www.promocell.com/cell-model-systems.

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Documents

Macrophage cell culture€¦ · Macrophage cell culture Fig. 1: PromoCell macrophage cell culture portfolio. Overview of possible applications with PromoCell's macrophage media and