title: Cell fixation in zinc salt solution is compatible with DNA damage response detection by phospho-specific antibodies.
abstract: By virtue of superior preservation of proteins and nucleic acids the zinc salt-based fixatives (ZBF) has been proposed as an alternative to precipitants and cross-linking fixatives in histopathology. It was recently reported that ZBF is compatible with analysis of cell surface immunophenotype and detection of intracellular epitopes by flow cytometry. The aim of this study was to explore whether ZBF is also compatible with the detection of DNA damage response assessed by phospho-specific antibodies (Abs) detecting phosphorylation of the key proteins of that pathway. DNA damage in human pulmonary adenocarcinoma A549 cells was induced by treatment with the DNA topoisomerase I inhibitor camptothecin and phosphorylation of histone H2AX on Ser139 (γH2AX) and of ATM on Ser1981 was detected with phospho-specific Abs; cellular fluorescence was measured by laser scanning cytometry (LSC). The sensitivity and accuracy of detection of H2AX and ATM phosphorylation concurrent with the detection of DNA replication by EdU incorporation and "click chemistry" was found in ZBF fixed cells to be comparable to that of cell fixed in formaldehyde. The accuracy of DNA content measurement as evident from the resolution of DNA content frequency histograms of cells stained with DAPI was somewhat better in ZBF- than in formaldehyde-fixed cells. The pattern of chromatin condensation revealed by the intensity of maximal pixel of DAPI that allows one to identify mitotic and immediately post-mitotic cells by LSC was preserved after ZBF fixation. ZBF fixation was also compatible with the detection of γH2AX foci considered to be the hallmarks of induction of DNA double-strand breaks. Analysis of cells by flow cytometry revealed that ZBF fixation of lymphoblastoid TK6 cells led to about 60 and 33% higher intensity of the side and forward light scatter, respectively, compared to formaldehyde fixed cells.
authors: Department of Pathology, Brander Cancer Research Institute, New York Medical College, Valhalla, New York 10595, USA.
authors: Hong
authors: H
authors: Zhao
authors: Jiangwei
authors: J
authors: Li
authors: Frank
authors: F
authors: Traganos
authors: H Dorota
authors: HD
authors: Halicka
authors: Mirosław
authors: M
authors: Zarebski
authors: Jurek
authors: J
authors: Dobrucki
authors: Zbigniew
authors: Z
authors: Darzynkiewicz
references: Cell Cycle. 2008 Oct;7(19):3048-55
references: 18802408
references: Cytometry A. 2011 May;79(5):328-37
references: 21425239
references: J Histochem Cytochem. 2010 Jan;58(1):29-39
references: 19755718
references: Cytometry A. 2007 Sep;71(9):648-61
references: 17622968
references: Pathol Res Pract. 2001;197(12):823-6
references: 11795830
references: Histochemistry. 1985;82(3):201-4
references: 3997553
references: Cytometry A. 2007 Nov;71(11):905-14
references: 17879239
references: Cytometry A. 2009 Jun;75(6):510-9
references: 19373929
references: J Histochem Cytochem. 2010 Mar;58(3):237-46
references: 19901271
references: Cytometry. 1996 Apr 1;23(4):272-8
references: 8900469
references: Cell. 2006 Jun 30;125(7):1297-308
references: 16814716
references: Cell Prolif. 2005 Aug;38(4):223-43
references: 16098182
references: Cytometry A. 2004 Apr;58(2):99-110
references: 15057963
references: Rom J Morphol Embryol. 2006;47(1):15-9
references: 16838052
references: Cytometry A. 2010 Nov;77(11):999-1007
references: 20939035
references: Lab Invest. 2003 Jun;83(6):889-99
references: 12808124
references: Cell Cycle. 2010 Aug 15;9(16):3218-26
references: 20724827
references: Biotech Histochem. 2006 Jan-Feb;81(1):23-30
references: 16760124
references: Exp Cell Res. 1998 Feb 25;239(1):104-10
references: 9511729
references: Cell Cycle. 2006 Sep;5(17):1940-5
references: 16940754
references: Cytometry A. 2010 Apr;77(4):399-405
references: 20131407
references: Radiat Res. 2002 Oct;158(4):486-92
references: 12236816
references: Cytometry A. 2010 Aug;77(8):798-804
references: 20653019
references: Exp Cell Res. 1991 Mar;193(1):27-35
references: 1995300
references: Cell. 2004 Jul 9;118(1):9-17
references: 15242640
references: Exp Hematol. 2006 Sep;34(9):1183-91
references: 16939811
references: Anal Biochem. 1994 May 1;218(2):314-9
references: 8074286
references: Micron. 2001 Oct;32(7):645-52
references: 11334733
references: Methods Mol Biol. 2006;319:165-92
references: 16719355
references: J Histochem Cytochem. 1994 Aug;42(8):1127-34
references: 8027531
references: Proc Natl Acad Sci U S A. 2008 Feb 19;105(7):2415-20
references: 18272492
references: Biotech Histochem. 1994 May;69(3):177-9
references: 8068812
references: Nucleic Acids Res. 2007;35(12):e85
references: 17576663
references: Exp Cell Res. 1996 Jan 10;222(1):28-37
references: 8549670