PMID- 11598949
OWN - NLM
STAT- MEDLINE
DCOM- 20011205
LR  - 20191105
IS  - 0196-4763 (Print)
IS  - 0196-4763 (Linking)
VI  - 45
IP  - 1
DP  - 2001 Sep 1
TI  - Bivariate analysis of cellular DNA versus RNA content by laser scanning cytometry 
      using the product of signal subtraction (differential fluorescence) as a separate 
      parameter.
PG  - 73-8
AB  - BACKGROUND: The cytometric methods of bivariate analysis of cellular RNA versus 
      DNA content have limitations. The method based on the use of metachromatic 
      fluorochrome acridine orange (AO) requires rigorous conditions of the equilibrium 
      staining whereas pyronin Y and Hoechst 33342 necessitate the use of an instrument 
      that provides two-laser excitation, including the ultraviolet (UV) light 
      wavelength. METHODS: Phytohemagglutinin (PHA)-stimulated human lymphocytes were 
      deposited on microscope slides and fixed. DNA and double-stranded (ds) RNA were 
      stained with propidium iodide (PI) and protein was stained with BODIPY 630/650-X 
      or fluorescein isothiocyanate (FITC). Cellular fluorescence was measured with a 
      laser scanning cytometer (LSC). The cells were treated with RNase A and their 
      fluorescence was measured again. The file-merge feature of the LSC was used to 
      record the cell PI fluorescence measurements prior to and after the RNase 
      treatment in list mode, as a single file. The integrated PI fluorescence 
      intensity of each cell after RNase treatment was subtracted from the fluorescence 
      intensity of the same cell measured prior to RNase treatment. This RNase-specific 
      differential value of fluorescence (differential fluorescence [DF]) was plotted 
      against the cell fluorescence measured after RNase treatment or against the 
      protein-associated BODIPY 630/650-X or FITC fluorescence. RESULTS: The 
      scattergrams were characteristic of the RNA versus DNA bivariate distributions 
      where DF represented cellular ds RNA content and fluorescence intensity of the 
      RNase-treated cells, their DNA content. The distributions were used to correlate 
      cellular ds RNA content with the cell cycle position or with protein content. 
      CONCLUSIONS: One advantage of this novel approach based on the recording and 
      plotting of DF is that only the RNase -specific fraction of cell fluorescence is 
      measured with no contribution of nonspecific components (e.g., due to the 
      emission spectrum overlap or stainability of other than RNA cell constituents). 
      Another advantage is the method's simplicity, which ensues from the use of a 
      single dye, the same illumination, and the same emission wavelength detection 
      sensor for measurement of both DNA and ds RNA. The method can be extended for 
      multiparameter analysis of cell populations stained with other fluorochromes of 
      the same-wavelength emission but targeted (e.g., immunocytochemically) for 
      different cell constituents.
CI  - Copyright 2001 Wiley-Liss, Inc.
FAU - Smolewski, P
AU  - Smolewski P
AD  - Brander Cancer Research Institute, New York Medical College, Valhalla, New York 
      10523, USA.
FAU - Grabarek, J
AU  - Grabarek J
FAU - Kamentsky, L A
AU  - Kamentsky LA
FAU - Darzynkiewicz, Z
AU  - Darzynkiewicz Z
LA  - eng
GR  - R01 CA028704/CA/NCI NIH HHS/United States
GR  - R01 CA028704-24/CA/NCI NIH HHS/United States
GR  - CA 28704/CA/NCI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Cytometry
JT  - Cytometry
JID - 8102328
RN  - 0 (Phytohemagglutinins)
RN  - 0 (Proteins)
RN  - 63231-63-0 (RNA)
RN  - 9007-49-2 (DNA)
SB  - IM
MH  - Cell Cycle/genetics
MH  - DNA/*analysis
MH  - Fluorescence
MH  - Humans
MH  - Image Cytometry/*methods
MH  - Lymphocyte Activation/drug effects
MH  - Lymphocytes/immunology
MH  - Microscopy, Confocal/*methods
MH  - Phytohemagglutinins/pharmacology
MH  - Proteins/analysis
MH  - RNA/*analysis
MH  - Staining and Labeling
EDAT- 2001/10/13 10:00
MHDA- 2002/01/05 10:01
CRDT- 2001/10/13 10:00
PHST- 2001/10/13 10:00 [pubmed]
PHST- 2002/01/05 10:01 [medline]
PHST- 2001/10/13 10:00 [entrez]
AID - 10.1002/1097-0320(20010901)45:1<73::AID-CYTO1146>3.0.CO;2-A [pii]
AID - 10.1002/1097-0320(20010901)45:1<73::aid-cyto1146>3.0.co;2-a [doi]
PST - ppublish
SO  - Cytometry. 2001 Sep 1;45(1):73-8. doi: 
      10.1002/1097-0320(20010901)45:1<73::aid-cyto1146>3.0.co;2-a.