Product: Alexa Fluor® 647 anti-mouse/human CD11b

Catalog No.: 101218

Nouraiz Ahmed, Mr. D-BSSE, ETH Zurich

Live Cell Imaging of Granulocyte and Macrophage Progenitors Alexa Fluor® 647 anti-mouse/human CD11b)
Overall:	Product Quality:	Ease of Use:
This antibody gives a high signal for granulocytes and macrophages along with their progenitors. However, I also witnessed some associated background in confocal microscope.

Experimental Design

Application:	(Immunofluorescence) Live cell imaging of granulocyte and macrophages and their 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) Appropriate volume of desired antibodies is added to the cells. 4) Antibodies are washed away and proceeded with FACS-based sorting of hematopoietic stem and progenitor cells. 5) Cells are added to the cell culture plate under permissive medium (SFEM + SCF, TPO, Epo, IL3, IL6) and 1:10,000 concentration of CD11b-Alexa647 is added
Results Summary:	In live cell culture, this antibody gives a specific signal with granulocytes, macrophages and their progenitors (GMPs-donu shaped) as mentioned in the figure. However, there is also some background associated with this antibody which might interfere with the accurate quantification of the signal, though following the fate of GMPs would be ideal in this case.
Additional Notes:	This antibody is quite specific for granulocytes, macrophages and their progenitors (GMPs). Adding this antibody directly into the cell culture medium also helps to minimize the laborious and time consuming steps of immunonoflourescence and also helps to follow the fate of granulocyte and macrophage progenitors using time lapse imaging. Additionally, this antibody also stays inside the cell culture medium for more than four days indicating the high stability inside culture conditions.

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