Immunostaining and FACS analysis for pluripotency markers
A
B CD
Isotype controls
Isotype controls
Nanog/SSEA4
10
10
5
5
104
103
102
104
103
102
103
104
105
SSEA4 Isotype Controls
106
102
102
103
104
SSEA4
105
106
Nanog/SSEA4
106
106
10
105
5
Nanog
106
Nanog Isotype Control
106
Nanog
Nanog Isotype Control
To determine the percentage of
pluripotent cells, iPSCs growing on
MEFs were rinsed and removed from
the dish and then separated into a
single-cell suspension using StemPro®
Accutase® Cell Dissociation Reagent
in preparation for use in the Attune®
Acoustic Focusing Cytometer. The cells
were then incubated for FACS analysis
with conjugated anti-Nanog and antiSSEA4 antibodies, as well as isotype
control antibodies. Nanog and SSEA4
are common markers of pluripotency,
and all clones were observed to be over
87% positive for Nanog and nearly 100%
positive for SSEA4. Representative FACS
data are shown in Figure 5.
104
103
102
104
103
102
103
104
105
106
102
102
SSEA4 Isotype Controls
103
104
105
106
SSEA4
Figure 5. Immunostaining and FACS analysis demonstrating that iPSCs from PD-1 donor (A and B)
or Ctrl-1 (C and D) express pluripotency markers. iPSCs (passage 11, A and B; passage 12, C and D)
were cultured in KSR-supplemented iPSC medium on MEFs for 4 days. Cells were harvested, fixed,
permeabilized, and co-stained with isotype control antibodies (A and C) and antibodies against Nanog and
SSEA4 (B and D). FACS analysis was performed using the Attune® Acoustic Focusing Cytometer. The iPSCs
are observed to be over 87% positive for Nanog and nearly 100% positive for SSEA4.
A Journey Through Innovation, The Discovery of Acoustic
Focusing & the Attune® Flow Cytometer
Instrument highlight—Attune® Acoustic Focusing Cytometer
The Attune® Acoustic Focusing Cytometer was used to evaluate the percentage of
pluripotent stem cells in the iPSC population. With acoustic focusing, sample rates
can be increased by over 10 times compared to hydrodynamic focusing, so utilizing the
Attune® cytometer for this study allowed for a smaller starting sample. Evaluation of
the pluripotent markers Nanog and SSEA4 on the iPSCs demonstrated the cells to be
over 87% positive for Nanog and nearly 100% positive for SSEA4.
17 Life Technologies | Parkinson's cell model
