The document summarizes an experiment analyzing CCND1 expression in four breast cancer cell lines (MCF7, SKBR3, MDA-MB-231, T47D) using mass cytometry. The primary antibody STJ31335 CCND1 Antibody was used at 4 μg/ml along with a secondary antibody 159Tb-IgG goat anti-rabbit. The cells were cultured, fixed with paraformaldehyde, and stained according to the described protocol before mass cytometry acquisition. A reviewer commented that additional experiments are needed to demonstrate the specificity of the CCND1 antibody.

1. Cell Line:
MCF7, SKBR3, MDA-MB-231,
T47D cell lines (ATCC)
Method of validation: Mass Cytometry
Primary Antibody:
STJ31335 CCND1 Antibody
(STJ92538)
Secondary Antibody 159
Tb-IgG goat anti-rabbit
Dilution ratio: Used at 4 µg/ml
Result
“The antibody can be used for mass cytometry analysis of CCND1 expression. The specificity of the antibody to
CCND1 should be shown in an additional experiment.“ - J. Wagner, University of Zurich
Treatment of materials:
The cells were cultured to 70%-80% plating density. For cell detachment, they were washed with pre-warmed PBS
(Gibco) and 1X TrypLETM (Thermo Fisher) was added for 5 min at 37°C. For cell fixation, 1.6% paraformaldehyde
(Thermo Fisher) was added for 10 minutes at room temperature. Then, the cells were pipetted off the plate and col-
lected in cold full growth medium to quench the paraformaldehyde. The cells were spun down and aliquoted in PBS
(Gibco) prior to storage at -80°C.
Protocol:
Fixed cells were washed once with 1x PBS / 0.05% bovine serum albumin / 2mM EDTA cell staining buffer (CSB) prior to
staining with the antibody (STJ92356, 4 µg/ml) in CSB for 45 min at 22°C. The cells were washed three times with CSB.
Then, the secondary antibody, a goat anti-rabbit 159
Tb-IgG (0.25 µg/ml), was applied in CSB for 45 min at 22°C. The cells
were washed with CSB and ddH20 prior to mass cytometry acquisition.
Antibody Customer Review:
STJ31335 CCND1 Antibody (STJ92538)
Antibody Rating:
Figure: Overlay histogram showing different
mammary epithelial cell lines stained with
STJ31335 and anti-rabbit 159Tb-IgG (orange
line) or anti-rabbit 159
Tb-IgG alone (blue line).