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COEBP Robotics Case Study CS-04
Quantification of Endoplasmic Reticulum and Golgi apparatus
in cell lines issued from different clones
A high content screening study on the Thermofisher Robot
Background
Transfected Chinese hamster ovary (CHO) cell lines are widely
used for the production of recombinant proteins.
In all cell line development processes, identification and
characterisation of “good producers” issued from clonal selection
would be greatly aided by some form of phenotype profiling.
Here we look at endoplasmic reticulum and Golgi quantification of
CHO cell lines using the Organelle-ID RGB™ Reagent kit from
Enzo Life Sciences on a high content platform, the Arrayscan VTi.
Method
The Organelle-ID RGB™ Reagent IV is a mixture of the following
cell-permeable dyes: a red fluorescent endoplasmic reticulum dye,
a green fluorescent lysosomal dye and a blue fluorescent nucleic
acid dye. The staining pattern arising from the combination of
these three dyes permits visualization of the individual target
organelles by fluorescence/confocal microscopy.
Adherent CHO cells were cultured for 24 hours prior to assay in
4titude, 96 well Vision plates. Cells were stained according to the
Organelle-ID RGB kit protocol.
Image acquisition was performed in triplicate wells on an
Arrayscan VTi with its Live Cell Chamber set at 37C + 5% CO2.
Detailed cell structure was visible at 40x magnification.
Figure 1.
Top image shows ER (green) and
Golgi (red).
Bottom image shows organelle
segmentation in the spot detector
analysis. For each segmented cell,
ER and Golgi fluorescence
brightness are recorded.
Centre of Excellence in Biopharmaceuticals, The Michael Smith building, Manchester University
Oxford Road, M13 9PT, United Kingdom
http://www.coebp.ls.manchester.ac.uk/
Results
ER and Golgi Brightness intensity was measured per cell and
expressed relative to individual nuclear size (in each case size
was defined in pixels).
Across the five cell lines examined a clear difference in the
intensity of ER and Golgi brightness was noted. One of the cell
lines (cell line 3) exhibited less than half the ER brightness and
40% less Golgi brightness than the most intensely stained cell
line.
Conclusions
The Arrayscan Spotdetector Bioapplication and the Organelle-ID
RGB™ Reagent kit from Enzo Lifesciences were used together in
a study to detect and quantify changes in ER and Golgi intensity in
different cell lines.
We found that, whilst there was no statistically significant
difference in nuclear size (by the Arrayscan, which mirrored similar
cell sizes as detected with a Cedex cell counter), changes in
brightness intensity for ER and Golgi could be used to distinguish
cell lines issued from the same parental line. The consequence of
this phenotype difference towards the ability of individual cell lines
to support, for example, recombinant gene expression presents
intriguing approaches facilitated by high-content screening.
Figure 2.
ER vs Golgi can be plotted as a 2-D
histogram for each well (top: Cell
line 1, bottom: Cell line 3)
Figure 3.
ER and Golgi intensity tested for 5 cell lines, 3 well replicates per well, 25 fields per well, averages gathered
from around 1000 cells per well.
Please inquire about your specific application
For further information, contact Dr Egor Zindy
Centre of Excellence in Biopharmaceuticals, The University of Manchester, Oxford Road,
Manchester M13 9PT, United Kingdom.
Tel: +44 (0) 161 275 5418
E-mail: [email protected]
http://www.coebp.ls.manchester.ac.uk/