Isolation and Characterization of Mouse Innate Lymphoid Cells

Timotheus Y.F. Halim1, Fumio Takei2

1 Genetics Graduate Program, College for Interdisciplinary Studies, University of British Columbia, Vancouver, British Columbia, 2 Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 3.25
DOI:  10.1002/0471142735.im0325s106
Online Posting Date:  August, 2014
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Innate lymphoid cells (ILCs) are rare populations of cytokine‐producing lymphocytes and are divided into three groups, namely ILC1, ILC2, and ILC3, based on the cytokines that they produce. They comprise less than 1% of lymphocytes in mucosal tissues and express no unique cell surface markers. Therefore, they can only be identified by combinations of multiple cell surface markers and further characterized by cytokine production in vitro. Thus, multicolor flow cytometry is the only reliable method to purify and characterize ILCs. Here we describe the methods for cell preparation, flow cytometric analysis, and purification of murine ILC2 and ILC3. Curr. Protoc. Immunol. 106:3.25.1‐3.25.13. © 2014 by John Wiley & Sons, Inc.

Keywords: innate lymphoid cells (ILC); group 2 innate lymphoid cells (ILC2); group 3 innate lymphoid cells (ILC3); Rora ; Rorct ; flow cytometry

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Leukocytes Containing Innate Lymphocyte (ILC) Populations
  • Basic Protocol 2: Flow Cytometric Analysis of Innate Lymphoid Cells (ILCs)
  • Basic Protocol 3: Characterization of ILC2 by Intracellular Cytokine Detection
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Isolation of Leukocytes Containing Innate Lymphocyte (ILC) Populations

  Materials
  • Mice
  • Lung digestion medium (see recipe)
  • RPMI‐1640 medium, serum free
  • 40% (v/v) Percoll (GE Healthcare) in RPMI‐1640 (GE Healthcare)
  • Ammonium chloride RBC lysis buffer (Stem Cell Technologies, cat. no. 07850)
  • Phosphate‐buffered saline (PBS; ) containing 2% fetal bovine serum (FBS)
  • PBS ( ) containing 1 mM EDTA
  • Intestine digestion medium (see recipe)
  • Dissection tools
  • 10‐cm‐diameter Petri dishes
  • Razor blade or scalpel
  • 15 and 50‐ml conical polypropylene tubes (e.g., BD Falcon)
  • Shaking incubator
  • 70‐µm cell strainer (Fisher Scientific, cat. no. 22363548)
  • 3‐ and 5‐ml syringes
  • Centrifuge
  • 18‐G blunt end needle
  • 21‐G needle
  • 1‐ to 2‐mm wire mesh strainer
  • Additional reagents and equipment for euthanasia (unit ), removal of lungs (unit ), counting cells ( ), lymph node removal (unit ), and preparation of bone marrow cells (unit )

Basic Protocol 2: Flow Cytometric Analysis of Innate Lymphoid Cells (ILCs)

  Materials
  • Spleen cell suspension (unit ) to be used as single color– staining control cells
  • Single‐cell suspension containing ILCs ( protocol 1)
  • FcR‐blocking reagent (Fc receptor block; eBioscience, cat. 14‐0161‐85)
  • Antibodies for staining (see Tables 3.25.1 to 3.25.3) dissolved in PBS containing 2% FBS
  • Phosphate‐buffered saline (PBS; ) containing 2% (v/v) FBS
  • 1 mg/ml propidium iodine (PI) in Hanks buffered salt solution (HBSS; ) or equivalent viability stain (optional)
  • 5‐ml test tubes
  • Centrifuge
  • 5 ml filter‐cap test tubes (BD Falcon, cat. no. 352235)
  • Multicolor flow cytometer or FACS sorter (minimum 6‐color)

Basic Protocol 3: Characterization of ILC2 by Intracellular Cytokine Detection

  Materials
  • Single‐cell suspension containing ILCs ( protocol 1)
  • Re‐stimulation medium (see recipe)
  • FcR‐blocking reagent (Fc receptor block; eBioscience, cat. 14‐0161‐85)
  • Phosphate‐buffered saline (PBS; ) containing 2% (v/v) fetal bovine serum (FBS)
  • Antibodies for staining (see Table 3.25.4 for bone marrow and Table 3.25.5 for lung)
  • Intracellular staining reagents (BD Biosciences Cytofix/Cytoperm kit, or equivalent)
  • Fixable viability stain (eBioscience eFluor 780 or eFluor 450, or equivalent; optional)
  • 24‐well tissue culture plate
  • 5‐ml test tube
  • 5‐ml filter‐cap test tubes
  • Multicolor flow cytometer (minimum, 7‐color)
Table 3.5.4   MaterialsBone Marrow Immature ILC2 Staining Panel aLung ILC2 Intracellular Staining Panel a

Fluorochrome
Tube name PerCP‐Cy5.5 APC APC‐eFluor 780 eFluor 450 V500 PE PE‐Cy7
Unstained
PerCP‐Cy5.5 CD19
APC CD19
APC‐eFluor 780 B220
eFluor 450 CD19
V500 CD45
PE CD19
PE‐Cy7 Sca‐1
Sample CD25 CD117 B220 Lineage CD45 CD127 Sca‐1
Fluorochrome
Tube name eFluor 780 FITC PerCP‐Cy5.5 eFluor 450 V500 PE APC (Intracellular)
eFluor 780 Viability
FITC CD19
PerCP‐Cy5.5 CD19
eFluor 450 B220
V500 CD45
PE CD19
APC CD19
FMO control Viability T1/ST2 CD25 Lineage CD45 CD127 Isotype
Sample Viability T1/ST2 CD25 Lineage CD45 CD127 IL‐5

 aThis table shows a recommended staining strategy to identify immature ILC2 in the bone marrow. FcR‐blocked spleen cells may be used for unstained and single‐color controls. For single‐color control, we recommend the same antibody as used for the sample, unless the marker is dim, in which case we recommend a marker highly expressed on an abundant population (i.e., CD19 or B220 using spleen cells). PI buffer is used for all samples to exclude nonviable cells. The sample row indicates the MAb target ligated to its fluorochrome listed in the columns. Lineage MAbs include: CD3ϵ, CD19, NK1.1, Gr‐1, Ter119, and CD11b. All MAbs were used at 1/250 dilution in PBS containing 2% FBS, although individual titration is highly recommended for best results.
Table 3.5.5   MaterialsBone Marrow Immature ILC2 Staining Panel aLung ILC2 Intracellular Staining Panel a

Fluorochrome
Tube name PerCP‐Cy5.5 APC APC‐eFluor 780 eFluor 450 V500 PE PE‐Cy7
Unstained
PerCP‐Cy5.5 CD19
APC CD19
APC‐eFluor 780 B220
eFluor 450 CD19
V500 CD45
PE CD19
PE‐Cy7 Sca‐1
Sample CD25 CD117 B220 Lineage CD45 CD127 Sca‐1
Fluorochrome
Tube name eFluor 780 FITC PerCP‐Cy5.5 eFluor 450 V500 PE APC (Intracellular)
eFluor 780 Viability
FITC CD19
PerCP‐Cy5.5 CD19
eFluor 450 B220
V500 CD45
PE CD19
APC CD19
FMO control Viability T1/ST2 CD25 Lineage CD45 CD127 Isotype
Sample Viability T1/ST2 CD25 Lineage CD45 CD127 IL‐5

 aThis table shows a recommended staining strategy to identify intracellular cytokine (IL‐5) production in lung ILC2. FcR‐blocked spleen cells may be used for unstained and single‐color controls. For single‐color control we recommend the same antibody as used for the sample, unless the marker is dim, in which case we recommend a marker highly expressed on an abundant population (i.e., CD19 or B220 using spleen cells). A fixable viability dye (eFluor 780) is used to exclude nonviable cells. The sample row indicates the MAb target ligated to its fluorochrome listed in the columns. Lineage MAbs include: CD3ϵ, CD19, NK1.1, Gr‐1, Ter119, and CD11b. All surface‐staining MAbs were used at 1/250 dilution in PBS containing 2% FBS, anti‐IL5 MAb and isotype control were used at 1/100 dilution. Although recommended concentrations are listed, individual titration of MAbs is highly recommended for best results.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Bernink, J.H. , Peters, C.P. , Munneke, M. , te Velde, A.A. , Meijer, S.L. , Weijer, K. , Hreggvidsottir, H.S. , Heinsbroek, S.E. , Legrand, N. , Buskens, C.J. , Bemelman, W.A. , Mjosberg, J.M. , and Spits, H. 2013. Human type 1 innate lymphoid cells accumulate in inflamed mucosal tissues. Nat. Immunol. 14:221‐229.
   Eberl, G. , Marmon, S. , Sunshine, M.J. , Rennert, P.D. , Choi, Y. , and Littman, D.R. 2004. An essential function for the nuclear receptor RORgamma(t) in the generation of fetal lymphoid tissue inducer cells. Nat. Immunol. 5:64‐73.
   Halim, T.Y. , Maclaren, A. , Romanish, M.T. , Gold, M.J. , McNagny, K.M. , and Takei, F. 2012. Retinoic‐Acid‐receptor‐related orphan nuclear receptor alpha is required for natural helper cell development and allergic inflammation. Immunity 37:463‐474.
   Klose, C.S. , Kiss, E.A. , Schwierzeck, V. , Ebert, K. , Hoyler, T. , d'Hargues, Y. , Goppert, N. , Croxford, A.L. , Waisman, A. , Tanriver, Y. , and Diefenbach, A. 2013. A T‐bet gradient controls the fate and function of CCR6(‐)RORgammat(+) innate lymphoid cells. Nature 494:261‐265.
   Spits, H. , Artis, D. , Colonna, M. , Diefenbach, A. , Di Santo, J.P. , Eberl, G. , Koyasu, S. , Locksley, R.M. , McKenzie, A.N. , Mebius, R.E. , Powrie, F. , and Vivier, E. 2013. Innate lymphoid cells: A proposal for uniform nomenclature. Nat. Rev. Immunol. 13:145‐149.
   Walker, J.A. , Barlow, J.L. , and McKenzie, A.N. 2013. Innate lymphoid cells: How did we miss them? Nat. Rev. Immunol. 13:75‐87.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library