Product: PerCP/Cyanine5.5 anti-mouse CD16/32

Catalog No.: 101324

Nouraiz Ahmed, Mr. D-BSSE, ETH Zurich

Isolation of GMPs (granulocyte and macrophage progenitors) by FACS based sorting (PerCP/Cyanine5.5 anti-mouse CD16/32)
Overall:	Product Quality:	Ease of Use:
CD16/32 - PerCp/Cyanine5.5 gives a much brighter signal as compared to CD16/32-APC-Cyanine7 and gives a good resolution of GMPs against other blood and immune progenitors without any risk of compensation.

Experimental Design

Application:	FACS (Isolation of GMPs (granulocyte and macrophage progenitors))
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 strepavidin 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) 2 µl of CD16/32-PerCp/Cyanine5.5 antibody is added per 10 million cells and stained for 90 min on ice along with other antibodies of interest (e.g. Sca1, c-Kit, CD34, CD48, CD150, Lin). 4)Antibodies are washed away and proceeded with FACS-based sorting of GMPs.
Results Summary:	CD16/32 - PerCP/Cyanine5.5 gives a strong signal with 488 nm laser and detected within 488 A detector in BD FACS ARIA III. When I gate the progenitos cells (Lin -ve, Sca1-ve, c-Kit+ve cells) against CD16/32-PerCP/Cyanine5.5 and CD34, it results in 3 separate populations, GMPs, CMPs and MEPs (as mentioned in Akashi et al., 2000). Also when I gate the progenitors against CD150 and CD16/32-PerCP/Cyanine5.5, I see a strong bulge of GMPs at the top of CD16/32 negative erythroid progenitors. Thus this antibody helps better sorting of GMPs (as mentioned in the figure).
Additional Notes:	I would prefer this antibody over Alexa 700 conjugates.Since the signal is also strong, low concentration of antibody is usually preferred. Unlike, CD16/32-APC-Cyanine7, this antibody has some compensation issues with BV 711 and BV 650 dyes. But this is not a major problem. Overall, I would recommend this antibody.

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