Itgax (alpha) Rat Monoclonal Antibody [Clone ID: 223H7]

Specifications

Product Data
Clone Name	223H7
Applications	FC
Recommended Dilution	Cell separation: 20 μl/2.5x107 cells (ready for use). Flow cytometry: 2 μg/2.5x106 cells. For details see protocol below.
Reactivities	Mouse
Host	Rat
Isotype	IgG2a
Clonality	Monoclonal
Immunogen	Murine DC from C57BL/6 mice
Specificity	This antibody reacts with mouse CD11c.
Formulation	PBS Label: Biotin State: Liquid Ig fraction Stabilizer: 1% BSA Preservative: 0.09% NaN3
Concentration	lot specific
Purification	Protein G agarose
Conjugation	Biotin
Storage	Store at 2-8 °C.
Stability	Shelf life: one year from despatch.
Database Link	Entrez Gene 16411 Mouse UniProt ID Q9QXH4
Background	The CD11c (integrin α X; ~150 kDa) glycoprotein non-covalently associates with CD18 (integrin β 2; ~95 kDa) to form the heterodimeric complement receptor type 4 ( CR4), which is involved in monocyte/granulocyte adhesion during inflammatory responses. The CD11c/CD18 receptor binds to CD54, iC3b and fibrinogen and plays a role in leukocyte adhesive interactions. CD11c/CD18 is also implicated in B cell proliferation and mediates B cell binding to fibrinogen. CD11c is commonly used as a marker for dendritic cells, but it is also expressed on macrophages, monocytes, granulocytes, NK cells, activated T and B lymphocytes and microglia.
Synonyms	ITGAX, Integrin alpha-X, Leu M5
Note	This product was originally produced by MBL International. Protocol: Cell separation using magnetic beads 1) Isolate single cell suspension from mouse spleen by standard preparation method. 2) Wash the cells twice with washing buffer [PBS containing 0.5% BSA and 2 mM EDTA]. 3) Resuspend the cells with washing buffer (1x10 8 cells/mL). 4) Add 20 μ L of anti-mouse CD16/32 antibody to the cell suspension for blocking FcR, and incubate for 15 minutes at 4 o C. 5) Add 2 μ g per 2.5x10 7 cells of Biotin labeled mouse CD11c (223H7) to the cell suspension, and incubate for 30 minutes at 4 o C. Remove supernatant by careful aspiration. 6) Add 1 mL of the washing buffer followed by centrifugation at 500 x g for 1 minute at 4 o C. Remove supernatant by careful aspiration. 7) Resuspend cell pellet and label with streptavidin or anti-biotin magnetic beads according to the manufacturer’s recommendation. 8) Wash the separated cells with washing buffer. 9) Add 20 μ L of 1:40 diluted anti-mouse CD16/32 antibody with washing buffer to the cell suspension, and incubate for 10 minutes at 4 o C. 10) Add 20 μ L of PE labeled mouse CD11c (10 μ g/ml). Mix well and incubate for 1 hour at 4 o C. 11) Add 1 mL of the washing buffer followed by centrifugation at 500 x g for 1 minute at room temperature. Remove supernatant by careful aspiration. 12) Resuspend the cells with 500 μ L of the washing buffer and analyze by a flow cytometer. (Positive control for Flow cytometry; mouse splenocyte) Flow cytometric analysis for adherent cells We usually use Fisher tubes or equivalents as reaction tubes for all steps after 2). 1) Detach the cells from culture dish by cell dissociation buffer. 2) Wash the cells 3 times with washing buffer [PBS containing 0.5% BSA and 2 mM EDTA]. 3) Resuspend the cells with washing buffer (5x10 6 cells/mL). 4) Add 50 μ L of the cell suspension into each tube, and centrifuge at 500 x g for 1 minute at 4 o C. Remove supernatant by careful aspiration. 5) Add 20 μ L of 1:40 diluted anti-mouse CD16/32 antibody with washing buffer to the cell suspension. Mix well, and incubate for 10 minutes at 4 o C. 6) Add 1 μ g of the Biotin labeled Mouse CD11c monoclonal antibody (223H7). Mix well, and incubate for 30 minutes at 4 o C. 7) Add 1 mL of the washing buffer followed by centrifugation at 500 x g for 1 minute at 4 o C. Remove supernatant by careful aspiration. 8) Add 30 μL of 1:40 diluted PE conjugated streptavidin with the washing buffer. Mix well and incubate for 15 minutes at 4 o C. 9) Add 1 mL of the washing buffer followed by centrifugation at 500 x g for 1 minute at room temperature. Remove supernatant by careful aspiration. 10) Resuspend the cells with 500 μ L of the washing buffer and analyze by a flow cytometer.
Reference Data