Cell Line: MCF7, SKBR3, MDA-MB-231, T47D cell lines (ATCC)

Method of validation: Mass Cytometry

Primary Antibody: STJ31335 CCND1 Antibody (STJ92538)

Secondary Antibody 159Tb-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 159Tb-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.

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Figure: Overlay histogram showing different

mammary epithelial cell lines stained with

STJ31335 and anti-rabbit 159Tb-IgG (orange

line) or anti-rabbit 159Tb-IgG alone (blue line).