Biomedical Engineering Reference
In-Depth Information
Fig. 3
Analysis of selected cell populations. In this example, HeLa were transfected with mCherry and the cells
were loaded for 45 min with Alexa-488-labeled Tf in a recycling experiment, fi xed, stained with Hoechst 33342
and DY-647 phalloidin, and imaged in the corresponding four channels (Nucl, Phal, mCherry, and Tf). The nuclei
and the phalloidin channels are used to identify nuclei (
green outlines
) and cells (
red outlines
). The signal of
mCherry is used to distinguish transfected (
blue outlines
) from untransfected cells (
red outlines
) for separate
quantitation of transfected and untransfected cells. The mCherry signal is intentionally overexposed in order to
visualize low expressing cells
In the examples for endocytosis and recycling in Fig.
2
, the mean
intensity of Tf fl uorescence per cell was averaged for each well,
and the mean values of three wells per time point are plotted.
3.7 Analysis
of Selected Cell
Populations
To analyze selected cell populations, two
Pipelines
are built, the
fi rst one to measure the parameters that are used to distinguish
between cell populations, such as the fl uorescence intensity of a
fl uorescent protein-tagged protein of interest transfected into the
cells. For the example shown in Fig.
3
, this is the intensity of a fl uo-
rescent reporter (mCherry) transfected together with the protein
of interest on the same plasmid. To distinguish between transfected
and untransfected cells, it is essential to have control wells without
transfection on the same microplate. Cells can also be classifi ed in
more than two categories, for example, to distinguish between
high and low expressing cells as well. The second
Pipeline
is built
to measure Tf intensity.
1. The same
Pipeline
as in
steps 1
-
6
from Subheading
3.6
is set up.
2. The
Measure Object Intensity
module is used to distinguish
cells based on their fl uorescence intensity, for example, by
transfecting the cells with a fl uorescent reporter protein like
mCherry.
3. The
Display Data On Image
module is used to display the sin-
gle cell data of a certain measurement directly on the image
that was measured during an ongoing analysis. The measure-
ment to be displayed (e.g., mean fl uorescence intensity), the
channel from which the measurement is taken, and the channel
on which the data will be displayed are selected.
