Microorganisms and Plant Cells
1. Goals
Bacteria, marine plankton or isolated plant cells frequently exhibit a strong natural autofluorescence from chlorophyll or other pigments e.g. phycobiliproteins.
The concept for the flow cytometric determination of functional or constituent parameters of such cells is to determine autofluorescence above 600nm simultaneously with the fluorescences of functional or other biochemical stains below 600nm ( Tab.1, Tab.2).
2. Practical Concepts
Tab.1
INDO1
for Ca2+
is collected in fluorescence channels F1 and F2 between 390-440nm and
500-600nm while natural chlorophyll fluorescence is determined in
fluorescence channel F3 in addition to forward (FSC) and sideward (SSC)
light scatter signals.
In case of
blue-excitation
(488nm), functional dye fluorescence of e.g:
Tab.2
FDA
esterase activity in viable cells
is collected between 510-540nm and 540-580nm in fluorescence channels F1
and F2. Chlorophyll fluorescence is again collected in fluorescence
channel F3.
Natural autofluorescence may also occur in fluorescence channels F1 or F2.
In this case, the added fluorescent dyes due to their brightness should
still give valuable information. The natural fluorescence of each cell
cluster in an unstained control sample has to be subtracted from the
observed fluorescence of the respective cell cluster in the stained sample.
In case of
UV-excitation
(350-370nm), fluorescence emission from
added functional dyes like:
ADB
for intracellular pH and
OPT
for intracellular glutathione and free protein SH-groups
DNA
DiOC6(3)
for transmembrane potential
R123
mitochondrial potential
DHR
sensitive H2O2/peroxidase activity
DCFH-DA
H2O2/peroxidase
HE
O2-general oxidation
R110 proteinase substrates
cysteine/serine proteinases, aminopeptidases
AO
DNA of viable and dead cells
as well as PI as DNA counterstain for dead cells in all
UV
or
blue
excited cell function assays
For problems or comments, please contact:
G.Valet, E-mail:
valet@biochem.mpg.de,
Max-Planck-Institut für Biochemie,
Am Klopferspitz 18a, D-82152 Martinsried, Germany,
Tel: +49/89/8578-2518, -2525, Fax: +49/89/8578-2563,
INTERNET address:
http://www.biochem.mpg.de/valet/cellbio.html
Last Update: Feb.14, 2000