CCR7 Antibody (NBP1-43795)(0.1mg)

The 4B12 antibody has been tested by flow cytometric analysis of C57BL/6, Balb/C and SJL/J splenocytes and thymocytes.

Important: Staining with the 4B12 monoclonal antibody requires different conditions than typically used for surface-antigen staining. Please use the protocol below. Moreover, we have found that staining at 37°C, rather than 4°C, results in brighter 4B12 staining, as well as better resolution between positive and negative populations. Please see the data above which demonstrates a comparison of staining at 4°C and 37°C. Staining with 4B12 at 37°C is not expected to interfere with co-staining other antigens, however this should be evaluated for individual experiments.

1. Prepare cell suspension as normal and block FcγIIIR/FcγIIR with 5 μg/million cells purified anti-mouse CD16/32 (cat. 14-0161) for 15 minutes on ice. If red blood cell lysis is carried out as part of cell preparation, ensure that fixatives are not present in the red blood cell lysis solution as this will eliminate 4B12 staining.



2. Without washing, add 1 μg/million cells 4B12 and incubate in a 37°C waterbath or at 4°C (please see notes above) for 0.5 hours.

3. Wash cells 1X with 3 mls of Flow Cytometry Staining Buffer (cat. 00-4222) and decant supernatant.

4. Analyze cells on flow cytometer or proceed with secondary staining on ice as normal.

Note: Co-staining mouse CCR7 with the 4B12 antibody and the CCR7 ligand CCL19-Fc (cat. 14-1972) may be difficult due to different binding conditions required for the antibody versus the ligand, and steric hindrance which may prevent co-staining of 4B12 and CCL19-Fc. Cross-blocking experiments have demonstrated that 4B12 binding is able to prevent the detectable binding of CCL19-Fc, however not the opposite. Furthermore, the correlation between 4B12 and CCL19-Fc staining may be difficult to predict due to the presence of unknown CCL19-Fc receptors in addition to CCR7.

The 4B12 antibody has been tested by flow cytometric analysis of C57BL/6, Balb/C and SJL/J splenocytes and thymocytes.

Important: Staining with the 4B12 monoclonal antibody requires different conditions than typically used for surface-antigen staining. Please use the protocol below. Moreover, we have found that staining at 37°C, rather than 4°C, results in brighter 4B12 staining, as well as better resolution between positive and negative populations. Please see the data above which demonstrates a comparison of staining at 4°C and 37°C. Staining with 4B12 at 37°C is not expected to interfere with co-staining other antigens, however this should be evaluated for individual experiments.

1. Prepare cell suspension as normal and block FcγIIIR/FcγIIR with 5 μg/million cells purified anti-mouse CD16/32 (cat. 14-0161) for 15 minutes on ice. If red blood cell lysis is carried out as part of cell preparation, ensure that fixatives are not present in the red blood cell lysis solution as this will eliminate 4B12 staining.



2. Without washing, add 1 μg/million cells 4B12 and incubate in a 37°C waterbath or at 4°C (please see notes above) for 0.5 hours.

3. Wash cells 1X with 3 mls of Flow Cytometry Staining Buffer (cat. 00-4222) and decant supernatant.

4. Analyze cells on flow cytometer or proceed with secondary staining on ice as normal.

Note: Co-staining mouse CCR7 with the 4B12 antibody and the CCR7 ligand CCL19-Fc (cat. 14-1972) may be difficult due to different binding conditions required for the antibody versus the ligand, and steric hindrance which may prevent co-staining of 4B12 and CCL19-Fc. Cross-blocking experiments have demonstrated that 4B12 binding is able to prevent the detectable binding of CCL19-Fc, however not the opposite. Furthermore, the correlation between 4B12 and CCL19-Fc staining may be difficult to predict due to the presence of unknown CCL19-Fc receptors in addition to CCR7.