title: Detecting apoptosis of leukocytes in mouse lymph nodes.
abstract: Although there are multiple methods for analyzing apoptosis in cultured cells, methodologies for analyzing apoptosis in vivo are sparse. In this protocol, we describe how to detect apoptosis of leukocytes in mouse lymph nodes (LNs) via the detection of apoptotic caspases. We have previously used this protocol to study factors that modulate dendritic cell (DC) survival in LNs; however, it can also be used to analyze other leukocytes that migrate to the LNs. DCs labeled with a fluorescent cell tracker are subcutaneously injected in the posterior footpads of mice. Once the labeled DCs reach the popliteal LN (PLN), the animals are intravenously injected with FLIVO, a permeant fluorescent reagent that selectively marks active caspases and consequently apoptotic cells. Explanted PLNs are then examined under a two-photon microscope to look for the presence of apoptotic cells among the DCs injected. The protocol requires 6-6.5 h for preparation and analysis plus an additional 34-40 h to allow apoptosis of the injected DCs in the PLN.
authors.affiliation: 1] Centro de Investigaciones Biológicas. Consejo Superior de Investigaciones Científicas, Madrid, Spain. [2].
authors.forename: Laura
authors.initials: L
authors.lastname: Gómez-Cabañas
authors.affiliation: 1] Centro de Investigaciones Biológicas. Consejo Superior de Investigaciones Científicas, Madrid, Spain. [2].
authors.forename: Cristina
authors.initials: C
authors.lastname: Delgado-Martín
authors.affiliation: Centro de Investigaciones Biológicas. Consejo Superior de Investigaciones Científicas, Madrid, Spain.
authors.forename: Pilar
authors.initials: P
authors.lastname: López-Cotarelo
authors.affiliation: Centro de Investigaciones Biológicas. Consejo Superior de Investigaciones Científicas, Madrid, Spain.
authors.forename: Cristina
authors.initials: C
authors.lastname: Escribano-Diaz
authors.affiliation: Centro de Microscopía y Citometría, Universidad Complutense, Madrid, Spain.
authors.forename: Luis M
authors.initials: LM
authors.lastname: Alonso-C
authors.affiliation: 1] Centro de Investigaciones Biológicas. Consejo Superior de Investigaciones Científicas, Madrid, Spain. [2].
authors.forename: Lorena
authors.initials: L
authors.lastname: Riol-Blanco
authors.affiliation: Centro de Investigaciones Biológicas. Consejo Superior de Investigaciones Científicas, Madrid, Spain.
authors.forename: José Luis
authors.initials: JL
authors.lastname: Rodríguez-Fernández
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000276:immunology*; D001562:Benzimidazoles; D061945:Caspase Inhibitors / Q000378:metabolism; D005456:Fluorescent Dyes / Q000008:administration & dosage / Q000378:metabolism; D007962:Leukocytes / Q000166:cytology* / Q000276:immunology; D008198:Lymph Nodes / Q000166:cytology* / Q000276:immunology; D051379:Mice; D008954:Models, Biological
chemical_list: D001562:Benzimidazoles; D061945:Caspase Inhibitors; D005456:Fluorescent Dyes; C017807:bisbenzimide ethoxide trihydrochloride
references.citation: Immunol Rev. 2007 Oct;219:118-42
references.pmid: 17850486
references.citation: Nature. 2004 Jan 8;427(6970):154-9
references.pmid: 14712275
references.citation: J Immunol. 2005 Apr 1;174(7):4070-80
references.pmid: 15778365
references.citation: Am J Pathol. 1998 Apr;152(4):885-8
references.pmid: 9546348
references.citation: J Pathol. 1993 May;170(1):1-8
references.pmid: 8326456
references.citation: Glycobiology. 2011 May;21(5):655-62
references.pmid: 21199821
references.citation: Mol Cell. 2002 Mar;9(3):459-70
references.pmid: 11931755
references.citation: Science. 2012 Jun 29;336(6089):1676-81
references.pmid: 22745423
references.citation: J Biol Chem. 1998 Dec 4;273(49):32608-13
references.pmid: 9829999
references.citation: Magn Reson Med. 2010 Nov;64(5):1510-9
references.pmid: 20593373
references.citation: J Immunol. 2010 Jul 15;185(2):795-802
references.pmid: 20601611
references.citation: Pathology. 2000 Aug;32(3):186-90
references.pmid: 10968392
references.citation: Curr Opin Immunol. 2007 Oct;19(5):516-21
references.pmid: 17644328
references.citation: J Pathol. 2008 Jan;214(2):190-8
references.pmid: 18161756
references.citation: Nat Rev Mol Cell Biol. 2008 Mar;9(3):231-41
references.pmid: 18073771
references.citation: Immunology. 2002 May;106(1):87-95
references.pmid: 11972636
references.citation: Nat Immunol. 2009 Jul;10(7):753-60
references.pmid: 19503105
references.citation: Lab Anim (NY). 2008 Jan;37(1):26-32
references.pmid: 18094699
references.citation: Am J Physiol Cell Physiol. 2003 May;284(5):C1309-18
references.pmid: 12676658
references.citation: Nat Protoc. 2007;2(11):2719-27
references.pmid: 18007607
references.citation: Methods Mol Biol. 2011;682:3-13
references.pmid: 21057916
references.citation: J Immunol. 2009 Nov 15;183(10):6282-95
references.pmid: 19841191
references.citation: J Immunol. 2002 Sep 15;169(6):3006-14
references.pmid: 12218115
references.citation: Exp Cell Res. 2000 Aug 25;259(1):308-13
references.pmid: 10942603
references.citation: Lab Anim (NY). 2011 May;40(5):155-60
references.pmid: 21508954
references.citation: Cytometry A. 2003 Sep;55(1):50-60
references.pmid: 12938188
references.citation: Circulation. 1999 Jun 1;99(21):2757-64
references.pmid: 10351969
references.citation: Trends Immunol. 2007 Aug;28(8):346-52
references.pmid: 17625969
references.citation: Am J Physiol Cell Physiol. 2002 Sep;283(3):C905-16
references.pmid: 12176747
references.citation: J Exp Med. 2003 Aug 18;198(4):615-21
references.pmid: 12925677
references.citation: Nat Immunol. 2004 Jun;5(6):583-9
references.pmid: 15133508
references.citation: J Virol. 2010 May;84(10):4866-77
references.pmid: 20200235
references.citation: Arch Pathol Lab Med. 1999 Jun;123(6):529-32
references.pmid: 10383807
references.citation: Nat Protoc. 2006;1(3):1458-61
references.pmid: 17406435
references.citation: Nat Protoc. 2009;4(9):1383-95
references.pmid: 19730422
references.citation: Nat Protoc. 2008;3(6):955-64
references.pmid: 18546596
references.citation: J Exp Med. 2009 Mar 16;206(3):549-59
references.pmid: 19237601
references.citation: Toxicol Pathol. 2006;34(5):409-24
references.pmid: 17067937
references.citation: J Cell Biol. 2011 Jul 25;194(2):209-27
references.pmid: 21768291
references.citation: Methods Mol Biol. 2002;203:289-99
references.pmid: 12073450
references.citation: Circulation. 2012 Apr 3;125(13):1652-63
references.pmid: 22388323
references.citation: Blood. 2007 May 15;109(10):4360-7
references.pmid: 17227827
references.citation: Hum Cell. 2002 Mar;15(1):1-12
references.pmid: 12126059
references.citation: J Biol Chem. 2011 Oct 28;286(43):37222-36
references.pmid: 21878648
references.citation: J Exp Med. 2012 Jun 4;209(6):1135-52
references.pmid: 22615127
references.citation: J Immunol. 2006 May 1;176(9):5153-9
references.pmid: 16621978