The broadest portfolio for your innate lymphoid cell research

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1 The broadest portfolio for your innate lymphoid cell research Tissue dissociation Cell separation Cell culture Flow cytometry

2 Innate lymphoid cells Overview Innate lymphoid cells comprise various subpopulations Innate lymphoid cells (ILCs) have important effector and regulatory functions in innate immunity and tissue remodeling. They are defined by two characteristics: They have a lymphoid origin and differentiate from a hematopoietic common lymphoid progenitor (CLP). They lack antigen-specific receptors and therefore do not require RAG proteins for development. ILCs are present throughout the body and enriched at mucosal surfaces including intestines, lung, skin, liver, adipose tissue, and mesenteric lymph nodes, in both human and mouse. ILCs can respond rapidly to tissue disturbances and pathogenic threats. These cells have a complex phenotype, so characterization by multiparameter flow cytometry is important for an accurate analysis. ILCs are divided into three groups, based on cytokine production patterns and the requirement of transcription factors for development and function: Group ILCs produce interferon gamma (IFN-γ) and rely on T-bet. Group 2 ILCs produce type 2 cytokines like IL-5 and IL-3 and rely on GATA3. Group 3 ILCs include lymphoid tissue inducer (LTi) cells, produce IL-7 and/or IL-22, and are dependent on RORγ (t). Miltenyi Biotec offers a comprehensive portfolio for the dissociation of various tissues and the subsequent magnetic separation, flow cytometry analysis, and culture of ILCs. Group 3 ILCs ILC3 CHILP ILC2 PLZF ILCp RORγ (t) GATA 3 Group 2 ILCs CLP αlp ILC NFIL3 Id2 Tox T-bet Group ILCs NKP cnk Eomes T-bet Figure : Development of ILCs. The common lymphoid progenitor (CLP) is the basis for the development of all ILCs and gives rise to a committed ILC precursor population (αlp). At this point, ILC development bifurcates: One pathway ultimately leads to the formation of conventional natural killer cells (cnk) via an NK cell precursors (NKP). The other pathway results in the development of all other ILCs through a so-called common helper innate lymphoid precursor (CHILP). An up-regulation of the transcription factors Id2, PLZF, and GATA-3 results in the conversion of CHILP into committed innate lymphoid cell precursors (ILCp), which subsequently can develop into ILCs and most group 2 and 3 ILCs. Development of the two members of group ILCs, i.e., cnk and ILC, differs in the requirement of transcription factors. The development of NKP into mature cnk is critically dependent on Eomes and also involves T-bet. In contrast, ILC development from ILCp is dependent on T-bet and does not involve Eomes. For details see references and 2. References. Fuchs, A. (26) Front. Immunol. 7: Lim, A.W.Y. and McKenzie, A.N.J. (25) Genes Immun. 6: Juelke, K. and Romagnani, C. (26) Curr. Opin. Immunol. 38: Hazenberg, M.D. and Spits H. (26) Blood 24: Cortez, V. et al. (25) Curr. Opin. Immunol. 32:

ILC isolation and analysis Overview Human ILCs Blood Skin Whole Skin Dissociation Kit Epidermis Dissociation Kit Legend MACS Sample Preparation MACS Cell Separation MACS Antibody MACS Cell culture

3 ILC isolation and analysis Overview Human ILCs Blood Skin Whole Skin Dissociation Kit Epidermis Dissociation Kit Legend MACS Sample Preparation MACS Cell Separation MACS Antibody MACS Cell culture and stimulation Not available Enrichment of ILCs: st step: Depletion of T cells using CD3 MicroBeads 2nd step: CD27 MicroBead Kit ILC CD27 ILC CD6 CD3 +/ / CD6 + CCR6 + NKp44 + NKp46 + Eomes +/ Group ILCs Group 2 ILCs Group 3 ILCs NK cell CD56 + CD6 +/ CD94 + IL-2Rβ + NKp46 + Eomes + IL-2 IL-5 IL-8 NK Cell Isolation Kit CD56 MicroBeads ILC2 Lin * CD6 + CRTH2 + ICOS + GATA3 + ILC2 Isolation Kit Lineage Cell Depletion Kit CD27 MicroBead Kit IL-25 IL-33 TSLP ILC3 fetal LTi CD6 + CCR6 + ILC3 NKp44 CD7 + CD6 + CCR6 + NKp44 RORγ (t) + IL-β IL-23 ILC3 NKp44 + CD7 + CD6 + CCR6 + NKp44 + RORγ (t) + IFN-γ IL-5 IL-3 IL-22 IL-7 (fetal LTi) Mouse ILCs Blood Lung Intestine Skin Liver Lung Dissociation Kit Lamina Propria Dissociation Kit Epidermis Dissociation Kit Liver Dissociation Kit Enrichment of ILCs: Depletion of T cells using CD3ε MicroBead Kit Group ILCs Group 2 ILCs Group 3 ILCs ILC NK. + NKp46 + CD49a + CD49b TRAIL + / CD69 + Eomes NK cell NK. + NKp46 + CD49a CD49b + TRAIL CD27 CD69 Eomes + NK Cell Isolation Kit Anti-NKp46 MicroBead Kit CD49b (DX5) MicroBead Kit ILC2 Lin ** CD25 + Sca + KLRG + ICOS + ST2 + GATA3 + Lineage Cell Depletion Kit ILC3 fetal LTi NK. NKp46 CD4 +/ CCR6 + CD7 + RORγ (t) + T-bet ILC3 NKp46 NK. NKp46 CCR6 +/ CD7 + NKG2D RORγ (t) + ILC3 NKp46 + NK. NKp46 + CCR6 CD7 + NKG2D + RORγ (t) + IL-2 IL-5 IL-8 IL-25 IL-33 TSLP IL-β IL-23 IFN-γ IL-5 IL-3 IL-22 IL-7 (fetal LTi) 3 * CD3, CD4, CD4, CD9, CD2, CD36, CD66b, CD23, CD235ab, CDa, CD33, FcεRla The charts on this page were compiled based on references and 3 5 (see p. 2). ** CD3ε, CD4, CD8α, CDc, CD9, CD5, TER9, F4/8, FcεRlα, Gr-

4 Human ILCs Product tables Sample preparation Target tissue Product Order no. Skin Epidermis Dissociation Kit Tumor Tumor Dissociation Kit Umbilical cord Umbilical Cord Dissociation Kit Skin Whole Skin Dissociation Kit Cell separation Product Separation strategy Order no. NK cells NK Cell Isolation Kit Depletion CD56 MicroBeads Positive selection Pre-enrichment of ILCs Lineage Cell Depletion Kit Depletion CD3 MicroBeads Positive selection 3-5- CD27 MicroBead Kit Positive selection Anti-Biotin MicroBeads Depletion or positive selection IL-22 Secretion Assay Cell Enrichment and Detection Kit (PE) Positive selection ILC2 ILC2 Isolation Kit Positive selection CD294 (CRTH2) MicroBead Kit Positive selection Lineage Cell Depletion Kit Depletion Cell culture Product Research grade Premium grade NK MACS CD Medium TexMACS Medium IL-2 IL-5 IL-33 TSLP IL-β IL-23 IL-25 Secretion assays and MACSPlex Assays Product Order no. MACSPlex Cytokine 2 Kit IFN-γ Secretion Assay Detection Kit FITC PE APC IL-5 Secretion Assay Detection Kit PE APC IL-3 Secretion Assay Detection Kit PE IL-7 Secretion Assay Detection Kit PE APC IL-22 Secretion Assay Detection Kit PE

Human ILCs Flow cytometry antibodies Antigen Clone VioBlue VioGreen FITC VioBright FITC Vio 55 VioBright 55 PE PE-Vio 65 PerCP PerCP-Vio 77 PE-Vio 77 APC APC-Vio 77 Biotin ILC surface markers CD3

5 Human ILCs Flow cytometry antibodies Antigen Clone VioBlue VioGreen FITC VioBright FITC Vio 55 VioBright 55 PE PE-Vio 65 PerCP PerCP-Vio 77 PE-Vio 77 APC APC-Vio 77 Biotin ILC surface markers CD3 Ber-ACT 8 CD7 A3C6E2 CD7 AC26 CD22 (IL-2Rβ) REA67 CD27 MB5-8C9 CD27 REA64 CD6 VEP3 CD6 REA423 CD6 9B8 CD6 REA63 CD96 (CCR6) REA9 CD27 (IL-7RA) REA29 CD278 (ICOS) REA92 CD294 BM6 CD294 REA598 CD335 (NKp46) 9E2 CD336 (NKp44) 2.29 CD337 (NKp3) AF29-4D2 CD56 AF2-7H3 CD56 REA96 CD7 CD7-6B7 CD94 REA3 IL-2R β2 REA333 NKp8 4A4.D Secreted factors GM-CSF BVD2-2C IFN-γ 45-5 IFN-γ REA6 IL-2 (p35/p7) REA2 IL-3 JES-5A2.2 IL-7A CZ8-23G IL-7F LN2-9C4 IL-7RC REA57 IL-22 REA466 IL-4 7A3-3 IL-5 JES-39D IL-9 MH9D 5

6 Human ILCs Flow cytometry antibodies Antigen Clone VioBlue VioGreen FITC VioBright FITC Vio 55 VioBright 55 PE PE-Vio 65 PerCP PerCP-Vio 77 PE-Vio 77 APC APC-Vio 77 Biotin Transcription factorss CD254 (RANKL) DN254 GATA3 REA74 RORγ (t) REA278 T-bet REA2 Lineage markers CDb M/ CDb REA592 CDb REA73 CDc MJ4-27G2 CDc REA68 CD23 AC45 CD4 REA599 CD6 VEP3 CD6 REA423 CD9 LT9 CD9 REA675 CDa HI49 CD2 LT2 CD235a (Glycophorin A) REA75 CD3 BW264/56 CD3 REA63 CD33 (BDCA-2) AC44 CD33 (BDCA-2) REA693 CD36 AC6 CD4 M-T32 CD4 M-T466 CD4 REA623 CD45 5B CD45 REA747 CD56 AF2-7H3 CD56 REA96 CD66b REA36 CD8 BW35/8 Fab CD8 REA734 FcεRlα CRA 6 For more information about MACS Antibodies and REAfinity Recombinant Antibodies visit For customized fillings or bulk requests of pure antibodies for mass cytometry please go to

Human ILCs Featured application Highly pure type 2 ILCs from human blood CD294 (CRTH2) + type 2 ILCs (ILC2s) can be isolated effectively with the ILC2 Isolation Kit, human.

The isolated CRTH2 + cells are highly pure as shown by flow cytometry using a variety of antibodies (fig. 2).

PBMCs are magnetically labeled with biotinylated antibodies against lineagepositive cells and Anti-Biotin MicroBeads. 2. Lineage-negative cells are enriched using two consecutive LS Columns.

CD294 (CRTH2)-PE ³ ² ¹ - - - ¹ ² ³ - ¹ ² ³ ³ Lineage markers-fitc Lineage-negative cells CD6-PE-Vio 77 ³ ² ¹ Isolated CD294 (CRTH2) + ILC2 ³ CD27-APC 3.

7 Human ILCs Featured application Highly pure type 2 ILCs from human blood CD294 (CRTH2) + type 2 ILCs (ILC2s) can be isolated effectively with the ILC2 Isolation Kit, human. Figure illustrates the basic principle of the MACS Separation procedure consisting of two steps, i.e., depletion of lineage-positive cells and positive selection of the CRTH2 + cells. The isolated CRTH2 + cells are highly pure as shown by flow cytometry using a variety of antibodies (fig. 2). A CD294 (CRTH2)-PE ³ ² ¹ Lineage markers-fitc B Before separation ¹ ² ³ - ¹ ² ³ CD6-PE-Vio 77 ³ ² ¹ CD27-APC. PBMCs are magnetically labeled with biotinylated antibodies against lineagepositive cells and Anti-Biotin MicroBeads. 2. Lineage-negative cells are enriched using two consecutive LS Columns. Labeled lineagepositive cells are retained on the column. CD294 (CRTH2)-PE ³ ² ¹ ¹ ² ³ - ¹ ² ³ ³ Lineage markers-fitc Lineage-negative cells CD6-PE-Vio 77 ³ ² ¹ Isolated CD294 (CRTH2) + ILC2 ³ CD27-APC 3. Enriched lineage-negative cells are magnetically labeled with a CD294 (CRTH2)-PE antibody and Anti-PE MicroBeads. CD294 (CRTH2)-PE ² ¹ ¹ ² ³ - ¹ ² ³ Lineage markers-fitc CD6-PE-Vio 77 ² ¹ CD27-APC 4. CRTH2 + ILC2s are isolated using two consecutive MS Columns. Figure 2: Type 2 innate lymphoid cells (ILC2) were isolated from peripheral blood mononuclear cells by using the ILC2 Isolation Kit, two LS and two MS Columns, a MidiMACS Separator, and a MiniMACS Separator. The cells were fluorescently stained with CD45-VioBlue and lineage markers (CD2, CD3, CD4, CD6, CD23, CD235a (Glycophorin A) conjugated to FITC, CD9 and CD56 conjugated to VioBright FITC), and CD294 (CRTH2)-PE (A), or CD6-PE-Vio 77 and CD27-APC (B) and analyzed by flow cytometry using the MACSQuant Analyzer. Cell debris and dead cells were excluded from analysis based on scatter signals and propidium iodide fluorescence. CRTH2 + ILC2 Biotinylated antibody PE-conjugated antibody Non-ILC2s Anti-Biotin or Anti-PE MicroBead Figure : Schematic outlining the protocol for the isolation of CRTH2 + ILCs from PBMCs. Initially the lineage-positive cells are depleted using two LS Columns. The CRTH2 + cells are then isolated from the lineagenegative cell fraction by positive selection using two consecutive MS columns. The steps involving the second LS and MS Columns for lineage cell depletion and positive selection of ILC2s, respectively, have been omitted from the schematic. 7

8 Mouse ILCs Product tables Sample preparation Target tissue Product Order no. Skin Epidermis Dissociation Kit Small intestine Lamina Propria Dissociation Kit Liver Liver Dissociation Kit Lung Lung Dissociation Kit Muscle Skeletal Muscle Dissociation Kit Spleen Spleen Dissociation Kit Tumor Tumor Dissociation Kit Various Multi Tissue Dissociation Kit 3--2 Various Multi Tissue Dissociation Kit Various Multi Tissue Dissociation Kit Cell separation Product Separation strategy Order no. NK cells NK Cell Isolation Kit II Depletion CD49b (DX5) MicroBeads Positive selection Anti-NKp46 + MicroBead Kit Positive selection Cell culture Product Research grade Premium grade IL-2 IL-5 IL-β IL-23 IL-25 IL-33 Cytokine Secretion Assays and MACSPlex Assays Product Order no. Lineage Cell Detection Cocktail-Biotin MACSPlex Cytokine Kit Mouse IFN-γ Secretion Assay Detection Kit PE APC Mouse IL-5 Secretion Assay Detection Kit PE APC Mouse IL-7 Secretion Assay Detection Kit PE APC Pre-enrichment of ILCs Lineage Cell Depletion Kit Depletion Direct Lineage Cell Depletion Kit Depletion CD3ε MicroBead Kit Positive selection CD5 (Ly-) MicroBeads Positive selection CD45R (B22) MicroBeads Positive selection CD7 MicroBeads Positive selection Anti-Ter-9 MicroBeads Positive selection Anti-Biotin MicroBeads Depletion or positive selection

Mouse ILCs Flow cytometry antibodies Antigen Clone VioBlue VioGreen FITC VioBright FITC Vio 55 VioBright 55 PE PE-Vio 65 PerCP PerCP-Vio 77 PE-Vio 77 APC APC-Vio 77 Biotin ILC surface markers CD3 2E7

9 Mouse ILCs Flow cytometry antibodies Antigen Clone VioBlue VioGreen FITC VioBright FITC Vio 55 VioBright 55 PE PE-Vio 65 PerCP PerCP-Vio 77 PE-Vio 77 APC APC-Vio 77 Biotin ILC surface markers CD3 2E7 CD7 3C CD22 TM-β CD27 A7R 34 CD27 REA68 CD83 (CXCR3) CXCR3-73 CD83 (CXCR3) REA724 CD96 (CCR6) REA277 CD25 7D4 CD25 REA568 CD278 7E.7G9 CD278 (ICOS) REA92 CD34 CX5 CD4 GK.5 CD4 REA64 CD69 H.2F3 CD9. His5 CD9.2 3-H2 KLRG 2F NK. PK36 NKp46 29A.4.9 Sca- D7 Sca- REA422 Secreted factors IFN-γ AN IFN-γ REA638 IL-7A TC-8H IL-4 BVD4-D IL-5 TRFK-5 IL-9 RM9A4 TNF-α MP6-XT22 TNF-α REA636 (continued on the back page) 9

Antigen Clone VioBlue VioGreen FITC VioBright FITC Vio 55 VioBright 55 PE PE-Vio 65 PerCP PerCP-Vio 77 PE-Vio 77 APC APC-Vio 77 Biotin Transcription factors CD278 (ICOS) REA92 EOMES REA6 GATA3 REA74

10 Antigen Clone VioBlue VioGreen FITC VioBright FITC Vio 55 VioBright 55 PE PE-Vio 65 PerCP PerCP-Vio 77 PE-Vio 77 APC APC-Vio 77 Biotin Transcription factors CD278 (ICOS) REA92 EOMES REA6 GATA3 REA74 RORγ (t) REA278 T-bet REA2 TOX REA473 Lineage markers CDc N48 CD9 6D5 CD3ε 45-2C CD3ε 7A2 CD3ε REA66 CD CD5 REA42 CD8a CD8a REA6 F4/8 REA26 FcεRIα MAR- Gr- RB6-8C5 NK. PK36 Ter-9 Ter-9 For more information about MACS Antibodies and REAfinity Recombinant Antibodies visit For customized fillings or bulk requests of pure antibodies for mass cytometry please go to Germany/Austria/ Switzerland Miltenyi Biotec GmbH Friedrich-Ebert-Straße Bergisch Gladbach Germany Phone Fax USA/Canada Miltenyi Biotec Inc. 233 Lindbergh Street Auburn, CA 9562, USA Phone 8 FOR MACS Phone Fax macs@miltenyibiotec.com Australia Miltenyi Biotec Australia Pty. Ltd. Unit 6 A, 2 Eden Park Drive Macquarie Park NSW 23 Australia Phone Fax macs@miltenyibiotec.com.au Benelux Miltenyi Biotec B.V. Schipholweg 68 H 236 XE Leiden The Netherlands macs@miltenyibiotec.nl Customer service The Netherlands Phone Fax 8 42 Customer service Belgium Phone Fax Customer service Luxembourg Phone Fax China Miltenyi Biotec Technology & Trading (Shanghai) Co., Ltd. Rooms 233 and 239 No. 39, Xianxia Road Changning District 25 Shanghai, P.R. China Phone Fax macs@miltenyibiotec.com.cn France Miltenyi Biotec SAS rue Mercoeur 75 Paris, France Phone Fax macs@miltenyibiotec.fr Italy Miltenyi Biotec S.r.l. Via Paolo Nanni Costa 433 Bologna Italy Phone Fax macs@miltenyibiotec.it Japan Miltenyi Biotec K.K. Nittsu-Eitai Building 5F 6- Fuyuki, Koto-ku, Tokyo 35-4, Japan Phone Fax macs@miltenyibiotec.jp Nordics and Baltics Miltenyi Biotec Norden AB Scheelevägen Lund Sweden macs@miltenyibiotec.se Customer service Sweden Phone 2-8 Fax Customer service Denmark Phone Fax Customer service Norway, Finland, Iceland, and Baltic countries Phone Fax Singapore Miltenyi Biotec Asia Pacific Pte Ltd. Beach Road #28-6 to 28-8 Shaw Tower Singapore 8972 Phone Fax macs@miltenyibiotec.com.sg South Korea Miltenyi Biotec Korea Co., Ltd Arigi Bldg. 8F 562 Nonhyeon-ro Gangnam-gu Seoul 636, South Korea Phone Fax macs@miltenyibiotec.co.kr Spain Miltenyi Biotec S.L. C/Luis Buñuel 2 Ciudad de la Imagen Pozuelo de Alarcón (Madrid) Spain Phone Fax macs@miltenyibiotec.es United Kingdom Miltenyi Biotec Ltd. Almac House, Church Lane Bisley, Surrey GU24 9DR, UK Phone Fax macs@miltenyibiotec.co.uk Miltenyi Biotec provides products and services worldwide. Visit to find your nearest Miltenyi Biotec contact. Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use. MACS, the MACS logo, MACSQuant, MidiMACS, MiniMACS, REAfinity, TexMACS, Vio, VioBlue, VioBright, and VioGreen are registered trademarks or trademarks of Miltenyi Biotec GmbH and/or its affiliates in various countries worldwide. Copyright 27 Miltenyi Biotec GmbH and/or its affiliates. All rights reserved

MACSxpress Technology Untouched cell isolation directly from blood products

MACSxpress Technology Untouched cell isolation directly from blood products MACSxpress Isolation Technology Fast untouched cell isolation in 25 min No density gradient centrifugation Flexible sample volumes