BD FACSAria III

Differentiating H9 cells

Differentiating H9 cells

This kit contains three different fluorescent antibodies that can be used to identify both undifferentiated (TRA-1-81 and SSEA-3) and differentiated (SSEA-1) pluripotent stem cells. This combination of markers has been widely used to characterize and isolate differentiated and undifferentiated stem cells derived from hESCs and iPS cells.

Gating strategy for sorting

Gating strategy used to sort CD45RA+ and CD45RA- Tregs

The BD FACSAria II system was set up for a sort using either a 70-μm or 100-μm nozzle (70 psi or 35 psi with a frequency of 87 or 60 kHz respectively). CD45RA+ Tregs and CD45RA- Tregs were sorted in purity mode at a rate of 10,000 to 11,000 events per second.

The 561-nm laser
The 561-nm laser excites mCherry and PE more efficiently compared to the 488-nm laser.
The same sample of stained mouse spleen cells was acquired using a BD FACSAria II (488-nm, 405-nm, and 633-nm lasers) and BD FACSAria III (488-nm, 405-nm, 633-nm, and 561-nm lasers). Dim staining populations can only be detected using the 561-nm laser.
Representative FoxP3 staining of sorted Tregs
Representative FoxP3 staining of sorted Tregs
To determine purity, as defined by FoxP3+ status, a portion of the cells was stained with anti-human FoxP3 BD Horizon™ V450. Data is representative of 10 experiments.
CFP-transfected HeLa cells
CFP-transfected HeLa cells
The 445-nm laser excites Cyan Fluorescent Protein (CFP) more efficiently compared to the 405-nm laser. The same sample of CFP-transfected HeLa cells was acquired on the BD FACSAria III and excited either by the 445-nm laser or by the 405-nm laser. The cells excited by the 445-nm laser demonstrated improved separation compared to those excited by the 405-nm laser.
Mouse bone marrow side population
Mouse bone marrow side population

Mouse bone marrow cells were stained with Hoechst 33342, c-Kit, Sca-1, and lineage markers, and run on the BD FACSAria III equipped with a 375-nm laser.

The SP fraction (red) was limited to those cells expressing c-Kit+ Sca-1++.

Antibodies used were c-Kit PE, Sca-1 PE-Cy™7, Lineage FITC, CD34 APC, CD45 APC-Cy7, PI, Hoechst Blue, and Hoechst Red.

Cancer Cell Line Side Population
Cancer cell line side population
Human HT-29 colon cancer cells were stained with Hoechst 33342 and acquired on the BD FACSAria III equipped with a 375-nm laser (left dot plot). As a control, side population expression was blocked (right dot plot).
Platform comparison using CD4
Platform comparison using CD4
Comparison of whole blood stained with single-color CD4 FITC, CD4 APC, and CD4 Pacific Blue™ and run on both the BD FACSAria II and BD FACSCanto™ II systems. The BD FACSAria II was set for high-speed sorting (70 psi and 90 kHz), and both instruments were set up using BD Cytometer Setup and Tracking software.