Product: Pacific Blue™ anti-mouse Ly-6A/E (Sca-1)

Catalog No.: 108120

Nouraiz Ahmed, Mr. D-BSSE, ETH Zurich

Isolation of Hematopoeitic Stem and Progenitor Cells by FACS Based Sorting (Pacific Blue™ anti-mouse Ly-6A/E (Sca-1))
Overall:	Product Quality:	Ease of Use:
Despite its low signal, Sca1-Pacific Blue permits a very good resolution, and does not interfere with any other antibody according to my experience, therefore, I would highly recommend this antibody.

Experimental Design

Application:	FACS (Isolation of hematopoeitic stem and progenitor cells)
Cells used:	Lineage depleted whole bone marrow cells from mouse
Brief Protocol:	1)Whole bone marrow cells are isolated from femur, tibia and spine of mouse. 2)lineage cells are depleted using streptavidin conjugated CD19, CD3e, B220, Gr1, Ter119 and CD11b antibodies (7 min incubation) followed by incubation with biotinylated magnetic beads (7 min incubation), which are removed by placing the cells in a magnet (7 min incubation). 3) 4 µl of Sca1-Pacific Blue antibody is added per 10 million cells and stained for 90 min on ice along with other antibodies of interest (e.g. CD150, c-Kit, CD34, CD48, CD16/32, Lin). 4)Antibodies are washed away and proceeded with FACS-based sorting of hematopoietic stem and progenitor cells.
Results Summary:	This antibody gives a weak signal with 405 nm laser and detected in 405 F detector in BD FACS ARIA III. When I gate lineage negative cells against c-Kit and Sca1-Pacific Blue, I can clearly distinguish between Sca1-high cells and Sca-1 low cells. Therefore, this antibody permits a nice resolution of hematopoeitic stem and progenitor cells (in the LSK gate) as mentioned in the figure.
Additional Notes:	This antibody has low signal, therefore, I would recommend using a high volume of this antibody. However, despite its low signal, this antibody is quite friendly in its nature, gives ideal resolution and does not interfere with any other antibody or channel. There are no compensation issues with this antibody also. When using a lot of Brilliant Violet dyes, I would recommend using Sca1-Pacific Blue antibody since it will not pose any compensation issues and will give nice resolution of progenitors.

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