Immunocytochemistry for pluripotency markers
In order to evaluate the expression of pluripotent markers
after at least 10 passages post–iPSC generation, the iPSCs
were passaged into 8-well chamber slides that were precoated with Geltrex® LDEV-Free hESC-Qualified Reduced
Growth Factor Basement Membrane Matrix diluted 1:100
with DMEM/F-12, and expanded. The expanded clones were
tested for the expression of known pluripotency markers by
immunocytochemistry, following the user instructions for
each antibody. All clones stained positive for the common
pluripotent surface markers SSEA4, Tra-1-81, and Tra-1-60,
and the transcription factor Oct4, confirming expression of
pluripotency markers. Representative images taken on the
EVOS® FLoid® Cell Imaging Station are shown in Figure 4.
Instrument highlight—
EVOS® FLoid® Cell
Imaging Station
Fluorescent images of the iPSCs
expressing pluripotency markers
were taken with the EVOS® FLoid®
Cell Imaging Station. The three
color channels plus phase allow
for easy visualization of multiplexed staining and image
merging. The iPSCs express Oct4, Tra-1-81, SSEA4, and
Tra-1-60, confirming pluripotency.
15 Life Technologies | Parkinson's cell model
A
B
C
D
Figure 4. Immunocytochemistry demonstrating that iPSCs from PD-3 (A and
B) or MSA donor (C and D) express pluripotency markers. iPSCs (passage 11,
A and B; passage 13, C and D) were cultured in iPSC medium supplemented with
KnockOut™ Serum Replacement (KSR) on Geltrex®-coated chamber slides for 4
days. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton®
X-100, and co-stained for Oct4 (red) and Tra-1-81 (green) (A, C) or SSEA4 (red)
and Tra-1-60 (green) (B, D) at 1:400 dilution. Images were taken using the EVOS®
FLoid® Cell Imaging Station. Hoechst 33342 DNA staining is in blue.
FLoid Cell Imaging Station
