In Situ Single-Cell Western Blot on Adherent Cell Culture.
electrophoresis
hydrogels
in situ western blot
protein phosphorylation
single-cell studies
Journal
Angewandte Chemie (International ed. in English)
ISSN: 1521-3773
Titre abrégé: Angew Chem Int Ed Engl
Pays: Germany
ID NLM: 0370543
Informations de publication
Date de publication:
23 09 2019
23 09 2019
Historique:
received:
04
06
2019
revised:
12
07
2019
pubmed:
8
8
2019
medline:
18
9
2020
entrez:
8
8
2019
Statut:
ppublish
Résumé
Integrating 2D culture of adherent mammalian cells with single-cell western blotting (in situ scWB) uses microfluidic design to eliminate the requirement for trypsin release of cells to suspension, prior to single-cell isolation and protein analysis. To assay HeLa cells from an attached starting state, we culture adherent cells in fibronectin-functionalized microwells formed in a thin layer of polyacrylamide gel. To integrate the culture, lysis, and assay workflow, we introduce a one-step copolymerization process that creates protein-decorated microwells. After single-cell culture, we lyse each cell in the microwell and perform western blotting on each resultant lysate. We observe cell spreading after overnight microwell-based culture. scWB reports increased phosphorylation of MAP kinases (ERK1/2, p38) under hypertonic conditions. We validate the in situ scWB with slab-gel western blot, while revealing cell-to-cell heterogeneity in stress responses.
Identifiants
pubmed: 31390130
doi: 10.1002/anie.201906920
pmc: PMC6759404
mid: NIHMS1045949
doi:
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Review
Langues
eng
Sous-ensembles de citation
IM
Pagination
13929-13934Subventions
Organisme : Japan Society for the Promotion of Science
ID : JP15KK0297
Pays : International
Informations de copyright
© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
Références
Proc Natl Acad Sci U S A. 2015 Oct 13;112(41):12705-10
pubmed: 26417073
Nat Methods. 2016 Apr 28;13(5):405-14
pubmed: 27123816
Am J Physiol Renal Physiol. 2005 Jul;289(1):F2-7
pubmed: 15951478
Nat Mater. 2015 Sep;14(9):951-60
pubmed: 26168347
Biochim Biophys Acta. 2016 Sep;1860(9):2037-52
pubmed: 27261090
Cell. 2006 Aug 25;126(4):677-89
pubmed: 16923388
Biochem J. 2010 Aug 1;429(3):403-17
pubmed: 20626350
Tissue Eng Part A. 2009 Jan;15(1):147-54
pubmed: 18673086
J Colloid Interface Sci. 1999 Feb 1;210(1):157-166
pubmed: 9924119
J Biol Chem. 1995 Mar 31;270(13):7420-6
pubmed: 7535770
Nat Cell Biol. 2011 Nov 13;13(12):1457-65
pubmed: 22081092
Trends Biotechnol. 2018 Jan;36(1):105-118
pubmed: 29126572
Nat Rev Genet. 2011 Nov 03;12(12):833-45
pubmed: 22048664
Proc Natl Acad Sci U S A. 2006 Oct 24;103(43):15739-46
pubmed: 17023540
BMC Bioinformatics. 2014 Dec 30;15:431
pubmed: 25547324
Lab Chip. 2017 Dec 5;17(24):4312-4323
pubmed: 29120467
Integr Biol (Camb). 2009 Dec;1(11-12):625-34
pubmed: 20027371
Proc Natl Acad Sci U S A. 1990 May;87(9):3579-83
pubmed: 2333303
Nat Rev Cancer. 2006 Jun;6(6):449-58
pubmed: 16723991
Int J Mol Sci. 2015 Aug 05;16(8):18149-84
pubmed: 26251901
Biophys J. 2005 Jul;89(1):676-89
pubmed: 15849250
J Biol Chem. 1985 Apr 10;260(7):4492-500
pubmed: 3920218
Adv Mater. 2014 Feb 26;26(8):1242-7
pubmed: 24323894
Cell Immunol. 2018 Aug;330:188-201
pubmed: 29482836
PLoS One. 2010 Mar 23;5(3):e9445
pubmed: 20351781
Curr Protoc Cell Biol. 2010 Jun;Chapter 10:Unit 10.16
pubmed: 20521229
Nat Commun. 2018 Jan 31;9(1):449
pubmed: 29386514
Am J Physiol Cell Physiol. 2008 Oct;295(4):C1037-44
pubmed: 18753317
Ther Drug Monit. 2015 Jun;37(3):389-94
pubmed: 25970509
J Mammary Gland Biol Neoplasia. 1999 Apr;4(2):183-91
pubmed: 10426397
Adv Mater. 2014 Jan 8;26(1):125-47
pubmed: 24227691
EMBO J. 2010 Aug 4;29(15):2501-14
pubmed: 20588253
Ann N Y Acad Sci. 1998 Jun 30;851:139-46
pubmed: 9668616
Nat Methods. 2014 Jul;11(7):749-55
pubmed: 24880876
Nat Commun. 2015 Sep 09;6:8129
pubmed: 26350361
BMB Rep. 2017 Mar;50(3):117-125
pubmed: 27998397
Nat Mater. 2014 Oct;13(10):979-87
pubmed: 25108614
J Neurosci Methods. 2007 May 15;162(1-2):255-63
pubmed: 17368788
Mol Cell Proteomics. 2015 Jun;14(6):1672-83
pubmed: 25755294
Nat Rev Cancer. 2017 Jan 27;17(2):93-115
pubmed: 28127048
Exp Eye Res. 2006 Apr;82(4):588-96
pubmed: 16202406
Lab Chip. 2015 Feb 7;15(3):726-34
pubmed: 25428717
Nat Methods. 2012 Jan 30;9(2):152-8
pubmed: 22290187
J Cell Sci. 2010 Dec 15;123(Pt 24):4310-20
pubmed: 21123621
Physiol Rev. 2007 Oct;87(4):1441-74
pubmed: 17928589
Curr Opin Biotechnol. 2006 Aug;17(4):406-14
pubmed: 16806894
Am J Physiol Cell Physiol. 2008 Apr;294(4):C1046-55
pubmed: 18272822
Biotechniques. 2005 Dec;39(6):847-51
pubmed: 16382902
Nat Commun. 2015 Jun 24;6:7513
pubmed: 26104298
Nat Methods. 2013 Apr;10(4):315-23
pubmed: 23435261
ACS Biomater Sci Eng. 2018 Jul 9;4(7):2340-2349
pubmed: 33435100
Cell. 2006 Nov 3;127(3):635-48
pubmed: 17081983
Nat Cell Biol. 2016 Dec 23;19(1):1-9
pubmed: 28008184
Sci Signal. 2019 Jan 22;12(565):
pubmed: 30670635
Front Immunol. 2019 Mar 20;10:535
pubmed: 30949179
Science. 2001 Sep 7;293(5536):1829-32
pubmed: 11546872
Eur J Biochem. 1995 Jun 1;230(2):416-23
pubmed: 7607210
Soft Matter. 2012 Jul 21;8(27):7197-7206
pubmed: 23002394
J Am Chem Soc. 2006 Mar 8;128(9):2768-9
pubmed: 16506733
Biophys J. 2004 Jan;86(1 Pt 1):617-28
pubmed: 14695306
J Cell Sci. 2004 Jul 15;117(Pt 16):3511-8
pubmed: 15226371
Clin Diagn Lab Immunol. 2002 Nov;9(6):1235-9
pubmed: 12414755
Biophys J. 2003 Nov;85(5):3329-35
pubmed: 14581234
Proc Natl Acad Sci U S A. 1997 Dec 9;94(25):13661-5
pubmed: 9391082
Soft Matter. 2007 Jan 23;3(2):168-177
pubmed: 32680260
Cell Transplant. 2017 Jun 9;26(6):1089-1102
pubmed: 28139195