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Bead Based Flow Cytometry
Assays for the Analysis of
Cellular Signaling
Randy Wetzel, Reginaldo Prioli, Andrew Romano and Bradley L. Smith • Cell Signaling Technology, Beverly MA 01915
PE
Streptavidin-PE
Biotin-Detection ab
Hist
1023
22
lysate
pHist
These results demonstrate that beads
labeled with activation state specific antibodies can be used to perform quantifiable
multi-parameter flow cytometric analyses of
cellular signaling from biological samples.
Events
0
M
1023
0
0
SS Lin
Phospho-Histone H3 (PE)
Detection of 3.2, 4.1, 5.7, and 6.7 um unconjugated
polystyrene beads (red, green, blue, and yellow,
respectively) by flow cytometry based on forward
and side scatter.
Phospho-Histone H3 (S10) bead-based analysis of
lysates from HeLa cells synchronized in G0- (blue),
G1/S- (magenta) or M-phase (green). Red = no
detection antibody
S6
Events
52
59
pS6
-
+
- PDGF
0
+
Anti-Rabbit lgG (PE)
Phospho-S6 Ribosomal Protein (PE)
Validation of S6 antibody-conjugated beads using
biotinylated anti-rabbit IgG antibody and streptavidin-PE
(green). Omission of anti-rabbit antibody (blue) or use
of unconjugated beads (red) abolished signal.
Phospho-S6 Ribosomal Protein (Ser235/236)
bead-based analysis of lysates from untreated
(blue) and PDGF-treated (green) NIH3T3 cells.
Red = no detection antibody
pAkt
Akt
71
7
6
5
+
-
+
-
PDGF
Normalized Fluorescence
CONCLUSIONS:
GS
Events
Polystyrene beads (Spherotech Inc.) were
conjugated with capture antibodies and
incubated with lysates from cancer cell lines
treated with agents that affect the phosphorylation state of target proteins. Captured
phospho proteins were labeled with biotinconjugated phospho-sensitive detection
antibodies and streptavidin-PE. The bead
complexes were then analyzed on an
FC500 flow cytometer (Beckman Coulter).
GO
M
4
3
2
1
0
METHODS:
GS
Events
The use of antibody-labeled beads in flow
cytometry allows for the multi-parametric
analysis of cell and tissue lysates. The addition of activation state-specific (eg. phospho-specific) antibodies extends this
method to the analysis of cell signaling in
cell lines and patient samples. In this
poster we describe the use of bead-based
flow cytometric assays to profile the cellular signaling events associated with a variety of cancer model systems.
GO
0
INTRODUCTION:
FS Lin
Capture ab beads
0
Phospho-Akt (PE)
Gab1
Phospho-Akt (S473) bead-based analysis of
lysates from untreated (blue) and PDGF-treated
(green) NIH3T3 cells. Red = no detection antibody
pCrkL
SHP-2
Bcr
BCR/ABL Bead-based analysis of lysates from K562 cells
using Bcr/Abl antibody-conjugated beads. Phosphorylated
Bcr and associated Gab1protein were detected. PhosphoCrkL and SHP-2 protein were not detected, associated with
active Bcr/Abl.
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