Product: APC/Cyanine7 anti-mouse CD16/32

Catalog No.: 101328

Nouraiz Ahmed, Mr D-BSSE, ETH Zurich

Isolation of GMPs (granulocyte and macrophage progenitors) by FACS based sorting (APC/Cyanine7 anti-mouse CD16/32)
Overall:	Product Quality:	Ease of Use:
CD16/32-APC-Cyanine7 works great. It gives a good resolution of Sca1-ve, c-Kit +ve progenitor cells against CD34 and CD16/32, thus helps to have high purity of GMPs after sorting.

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-APC-Cyanine7 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:	When I plot Lin -ve, Sca1-ve, c-kit+ve progenitor cells against CD34 and CD16/32 (APC-Cyanine7), the CD16/32-APC-Cyanine7 gives a very clear signal and I find a progenitor population which is double positive for CD34 and CD16/32 which represent the so called GMPs according to Akashi et al., 2000.
Additional Notes:	When Lin -ve, Sca1-ve, c-kit+ve progenitor cells are gated against CD34 and CD16/32 (APC-Cyanine7), the antibody signal is very bright, divided the progenitors into three different populations and thus, permits a high resolution of GMPs, CMPs and MEPs. I prefer to use 2 µl of this antibody per 10 million of bone marrow cells. However, additional volume could also be used to enhance signal but this would not impart further resolution. Additionally, this antibody does not spill over (or very little) into other detectors, and thus there are no issues of compensation.

Data Image Click on the image for full size data.

Publication Status This data has not been published.

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