Tyramide signal amplification for analysis of kinase activity by intracellular flow cytometry.
Intracellular flow cytometry permits quantitation of diverse molecular targets at the single-cell level. However, limitations in detection sensitivity inherently restrict the method, sometimes resulting in the inability to measure proteins of very low abundance or to differentiate cells expressing subtly different protein concentrations. To improve these measurements, an enzymatic amplification approach called tyramide signal amplification (TSA) was optimized for assessment of intracellular kinase cascades. First, Pacific Blue, Pacific Orange, and Alexa Fluor 488 tyramide reporters were shown to exhibit low nonspecific binding in permeabilized cells. Next, the effects of antibody concentration, tyramide concentration, and reaction time on assay resolution were characterized. Use of optimized TSA resulted in a 10-fold or greater improvement in measurement resolution of endogenous Erk and Stat cell signaling pathways relative to standard, nonamplified detection. TSA also enhanced assay sensitivity and, in conjunction with fluorescent cell barcoding, improved assay performance according to a metric used to evaluate high-throughput drug screens. TSA was used to profile Stat1 phosphorylation in primary immune system cells, which revealed heterogeneity in various populations, including CD4+ FoxP3+ regulatory T cells. We anticipate the approach will be broadly applicable to intracellular flow cytometry assays with low signal-to-noise ratios.
Clutter
Matthew R
MR
Department of Microbiology and Immunology, Baxter Laboratory for Stem Cell Biology, Stanford University School of Medicine, Stanford, California, USA.
Heffner
Garrett C
GC
Krutzik
Peter O
PO
Sachen
Kacey L
KL
Nolan
Garry P
GP
D000818:Animals; D045744:Cell Line, Tumor; D002469:Cell Separation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000032:analysis / Q000378:metabolism*; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D015850:Interleukin-6 / Q000494:pharmacology; D019169:Jurkat Cells; D007938:Leukemia / Q000188:drug therapy / Q000378:metabolism; D057230:Limit of Detection; D008223:Lymphoma / Q000188:drug therapy / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010750:Phosphoproteins; D010766:Phosphorylation; D011485:Protein Binding; D013154:Spleen / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods; D014439:Tyramine / Q000032:analysis / Q000737:chemistry*; D020298:U937 Cells
D015850:Interleukin-6; D010750:Phosphoproteins; D007371:Interferon-gamma; D048049:Extracellular Signal-Regulated MAP Kinases; D014439:Tyramine
J Histochem Cytochem. 1992 Oct;40(10):1457-63
1527370
Cytometry A. 2003 Aug;54(2):75-88
12879454
Annu Rev Pharmacol Toxicol. 1990;30:1-45
2111654
J Immunol Methods. 1989 Dec 20;125(1-2):279-85
2558138
J Immunol Methods. 2003 Jul;278(1-2):221-34
12957410
Curr Protoc Cytom. 2007 Apr;Chapter 1:Unit1.20
18770846
Cytometry B Clin Cytom. 2010 Mar;78(2):115-22
20014315
J Immunol Methods. 1999 Nov 19;230(1-2):113-20
10594358
Ann N Y Acad Sci. 1975 Jun 30;254:163-71
52308
Methods Cell Biol. 1994;41:351-76
7532265
Nat Protoc. 2006;1(3):1507-16
17406442
J Biomol Screen. 1999;4(2):67-73
10838414
Proc Natl Acad Sci U S A. 2001 Sep 11;98(19):10805-10
11535838
Cytometry A. 2003 Oct;55(2):61-70
14505311
Cytometry A. 2008 Sep;73(9):799-807
18548611
J Immunol. 2005 Aug 15;175(4):2366-73
16081807
Cytometry. 1996 Jan 1;23(1):48-53
14650440
Cell. 2004 Jul 23;118(2):217-28
15260991
J Immunol. 2005 Aug 15;175(4):2357-65
16081806
Cytometry. 2000 May 1;40(1):81-5
10754521
Nat Chem Biol. 2008 Feb;4(2):132-42
18157122
J Histochem Cytochem. 1995 Apr;43(4):347-52
7897179
Proc Natl Acad Sci U S A. 2009 Aug 4;106(31):12885-90
19433802
J Biol Chem. 1959 Jun;234(6):1611-4
13654426
Cytometry A. 2004 Mar;58(1):21-32
14994216
Cytometry. 2001 Apr 15;46(2):72-8
11309815
Am J Clin Pathol. 2001 Sep;116(3):429-36
11554172
Science. 2005 Apr 22;308(5721):523-9
15845847
J Immunol. 2006 Aug 1;177(3):1581-9
16849466
Curr Opin Mol Ther. 2006 Jun;8(3):215-24
16774041
J Histochem Cytochem. 1999 Apr;47(4):421-30
10082744
Clin Immunol. 2004 Mar;110(3):206-21
15047199
Drug Discov Today Technol. 2005 Autumn;2(3):295-302
24981951
Cytometry B Clin Cytom. 2009 Nov;76(6):355-64
19575390
Proc Natl Acad Sci U S A. 2001 Nov 20;98(24):13850-3
11707593
Cytometry A. 2005;63(1):1-9
15619730
Curr Opin Chem Biol. 2006 Feb;10(1):20-7
16406766
Am J Clin Pathol. 2006 Feb;125(2):241-50
16393687
J Allergy Clin Immunol. 2007 Nov;120(5):1208-15
17919707
Curr Protoc Immunol. 2007 Aug;Chapter 8:Unit 8.17
18432997
J Immunol Methods. 1987 May 4;99(1):53-8
2952733
Clin Chem. 1985 Sep;31(9):1481-6
3896568
Nat Methods. 2006 May;3(5):361-8
16628206
Ann N Y Acad Sci. 1993 Mar 20;677:28-39
8388180
Nat Biotechnol. 2002 Feb;20(2):155-62
11821861
J Histochem Cytochem. 1998 Jun;46(6):771-7
9603790