PMID- 9725553
OWN - NLM
STAT- MEDLINE
DCOM- 19981214
LR  - 20191102
IS  - 0196-4763 (Print)
IS  - 0196-4763 (Linking)
VI  - 33
IP  - 1
DP  - 1998 Sep 1
TI  - Enzyme kinetic reactions and fluorochrome uptake rates measured in individual 
      cells by laser scanning cytometry.
PG  - 1-9
AB  - This study was designed to explore the utility of a microscope-based 
      cytofluorometer, the laser scanning cytometer (LSC), for time-resolved kinetic 
      measurements. This instrument measures fluorescence of individual cells rapidly, 
      with high sensitivity and accuracy. Also recorded in a list mode fashion are the 
      spatial X-Y coordinates of the cell on the slide as well as the time of 
      individual cell measurement. Repeated measurement of each of a group of cells 
      within a selected area of the slide, thus, yields information on their 
      fluorescence parameters (integrated value, maximal pixel intensity, or 
      fluorescence area) as a function of time. Using the fluorogenic substrate 
      di-(leucyl)-rhodamine 110, we measured the kinetic activity of L-aminopeptidase 
      in HL-60 cells and in monocytes, granulocytes, and lymphocytes from human blood. 
      Likewise, the rate of fluorescein diacetate (FDA) hydrolysis by esterases was 
      measured in HL-60 cells. Also studied was the rate of uptake of the 
      lysosomo-trophic fluorochrome acridine orange (AO) by human leukocytes. Several 
      hundred cells per sample were measured rapidly with a time resolution of 20-60 s. 
      The resolution was inversely proportional to the number of cells within the 
      measured population. The kinetic curves constructed for individual cells had 
      clearly defined slope and plateau regions. During data analysis the kinetic plots 
      were matched with the respective cells; the latter were identified by their 
      position on the slide and classified by their fluorescence or image after 
      staining with Giemsa. Great intercellular variability was noted in enzyme 
      kinetics among cells of the same type, and differences were seen in rate of AO 
      uptake between granulocytes, monocytes, and lymphocytes. Fluorescence fading and 
      recovery was observed especially in the case of FDA hydrolysis and AO uptake. Our 
      results indicate that LSC can be used to rapidly measure the rate of uptake or 
      accumulation of a particular fluorochrome or the kinetics of enzymatic reactions 
      in individual cells of large populations to reveal intercellular variability or 
      the presence of a cell subpopulation with different kinetic properties.
FAU - Bedner, E
AU  - Bedner E
AD  - Brander Cancer Research Institute, Department of Pathology, New York Medical 
      College, Valhalla 10523, USA.
FAU - Melamed, M R
AU  - Melamed MR
FAU - Darzynkiewicz, Z
AU  - Darzynkiewicz Z
LA  - eng
GR  - R01 CA028704/CA/NCI NIH HHS/United States
GR  - R01 CA028704-20/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 (Enzymes)
RN  - 0 (Fluoresceins)
RN  - 0 (Rhodamines)
RN  - EC 3.1.- (Esterases)
RN  - F30N4O6XVV (Acridine Orange)
RN  - YL39R93PRE (diacetylfluorescein)
SB  - IM
MH  - Acridine Orange/metabolism
MH  - Enzymes/*metabolism
MH  - Esterases/metabolism
MH  - Flow Cytometry/*methods
MH  - Fluoresceins/metabolism
MH  - HL-60 Cells
MH  - Humans
MH  - Kinetics
MH  - *Lasers
MH  - Rhodamines/metabolism
EDAT- 1998/09/02 05:29
MHDA- 2000/06/20 09:00
CRDT- 1998/09/02 05:29
PHST- 1998/09/02 05:29 [pubmed]
PHST- 2000/06/20 09:00 [medline]
PHST- 1998/09/02 05:29 [entrez]
AID - 10.1002/(SICI)1097-0320(19980901)33:1<1::AID-CYTO1>3.0.CO;2-P [pii]
AID - 10.1002/(sici)1097-0320(19980901)33:1<1::aid-cyto1>3.0.co;2-p [doi]
PST - ppublish
SO  - Cytometry. 1998 Sep 1;33(1):1-9. doi: 
      10.1002/(sici)1097-0320(19980901)33:1<1::aid-cyto1>3.0.co;2-p.