Workflow for high-dimensional flow cytometry analysis of T cells from tumor metastases.
Journal
Life science alliance
ISSN: 2575-1077
Titre abrégé: Life Sci Alliance
Pays: United States
ID NLM: 101728869
Informations de publication
Date de publication:
10 2022
10 2022
Historique:
received:
24
11
2021
revised:
24
05
2022
accepted:
25
05
2022
entrez:
20
6
2022
pubmed:
21
6
2022
medline:
23
6
2022
Statut:
epublish
Résumé
We describe a multi-step high-dimensional (HD) flow cytometry workflow for the deep phenotypic characterization of T cells infiltrating metastatic tumor lesions in the liver, particularly derived from colorectal cancer (CRC-LM). First, we applied a novel flow cytometer setting approach based on single positive cells rather than fluorescent beads, resulting in optimal sensitivity when compared with previously published protocols. Second, we set up a 26-color based antibody panel designed to assess the functional state of both conventional T-cell subsets and unconventional invariant natural killer T, mucosal associated invariant T, and gamma delta T (γδT)-cell populations, which are abundant in the liver. Third, the dissociation of the CRC-LM samples was accurately tuned to preserve both the viability and antigenic integrity of the stained cells. This combined procedure permitted the optimal capturing of the phenotypic complexity of T cells infiltrating CRC-LM. Hence, this study provides a robust tool for high-dimensional flow cytometry analysis of complex T-cell populations, which could be adapted to characterize other relevant pathological tissues.
Identifiants
pubmed: 35724271
pii: 5/10/e202101316
doi: 10.26508/lsa.202101316
pmc: PMC9166301
pii:
doi:
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Informations de copyright
© 2022 Faccani et al.
Références
Nat Med. 2021 Feb;27(2):212-224
pubmed: 33574607
Liver Int. 2021 Apr;41(4):764-776
pubmed: 33548061
Nat Immunol. 2010 Mar;11(3):197-206
pubmed: 20139988
Immunol Rev. 2020 Nov;298(1):198-217
pubmed: 32840001
Cancer Sci. 2019 Jul;110(7):2080-2089
pubmed: 31102428
Nat Med. 2017 Jan 6;23(1):18-27
pubmed: 28060797
Nat Rev Immunol. 2016 Jul;16(7):449-62
pubmed: 27320317
Front Immunol. 2019 Jul 03;10:1515
pubmed: 31354705
Nat Rev Immunol. 2019 Nov;19(11):665-674
pubmed: 31570879
Front Immunol. 2017 Jun 30;8:761
pubmed: 28713381
Transl Oncol. 2021 Oct;14(10):101174
pubmed: 34243011
Anal Biochem. 2021 Aug 15;627:114210
pubmed: 34033799
Nat Commun. 2022 Mar 16;13(1):1372
pubmed: 35296658
Bioinformatics. 2016 Aug 15;32(16):2473-80
pubmed: 27153628
Nat Immunol. 2021 Oct;22(10):1190-1197
pubmed: 34489590
J Hematol Oncol. 2018 Mar 15;11(1):39
pubmed: 29544515
Nat Commun. 2019 Mar 25;10(1):1065
pubmed: 30911002
Immunol Cell Biol. 2018 May;96(5):507-525
pubmed: 29437263
Tissue Antigens. 2014 Apr;83(4):237-46
pubmed: 24641502
Blood. 2013 Jan 24;121(4):573-84
pubmed: 23160470
Immunity. 2016 Nov 15;45(5):1135-1147
pubmed: 27851914
Nat Protoc. 2012 Dec;7(12):2067-79
pubmed: 23138348
Nat Biotechnol. 2011 Oct 02;29(10):886-91
pubmed: 21964415
Methods. 2015 Feb;73:79-89
pubmed: 25449901
J Clin Invest. 2021 Aug 16;131(16):
pubmed: 34228641
Blood. 2017 Jun 29;129(26):3440-3451
pubmed: 28465341
Nat Rev Cancer. 2021 Jun;21(6):345-359
pubmed: 33837297
Semin Immunol. 2020 Apr;48:101406
pubmed: 33248905
AIDS Res Hum Retroviruses. 2017 Nov;33(S1):S31-S39
pubmed: 28882052
Curr Protoc Cytom. 2017 Jan 5;79:1.3.1-1.3.27
pubmed: 28055116
Cytometry A. 2015 Jul;87(7):636-45
pubmed: 25573116
Curr Protoc Immunol. 2017 Apr 3;117:5.4.1-5.4.38
pubmed: 28369683
Nat Rev Immunol. 2015 Aug;15(8):486-99
pubmed: 26205583
Annu Rev Immunol. 2016 May 20;34:479-510
pubmed: 26927205
Cell Rep. 2020 Dec 29;33(13):108571
pubmed: 33378668
J Clin Invest. 2021 Oct 1;131(19):
pubmed: 34411002
Eur J Immunol. 2017 May;47(5):765-779
pubmed: 28393361
BMC Bioinformatics. 2010 Nov 04;11:546
pubmed: 21050468
Nat Protoc. 2019 Jul;14(7):1946-1969
pubmed: 31160786
Front Immunol. 2021 Feb 16;11:619954
pubmed: 33664732
J Hepatol. 2018 Sep;69(3):654-665
pubmed: 29758330
Eur J Immunol. 2019 Oct;49(10):1457-1973
pubmed: 31633216
J Immunol Methods. 2019 Dec;475:112618
pubmed: 31181212
Eur J Immunol. 2021 Aug;51(8):1992-2005
pubmed: 34081326
Nat Rev Cancer. 2017 May;17(5):302-317
pubmed: 28303905
Nat Immunol. 2015 Nov;16(11):1114-23
pubmed: 26482978
Cell Rep. 2018 Mar 13;22(11):3006-3020
pubmed: 29539427
Curr Protoc Cytom. 2005 May;Chapter 1:Unit 1.3
pubmed: 18770811