CD197 (CCR7), APC-eFluor 780, clone: 4B12, eBioscience™
Rat Monoclonal Antibody
Brand: Affymetrix eBioscience 47-1971-80
Code : NEW
Additional Details : Weight : 0.09500kg
DescriptionCCR7 also plays a role in thymocyte development and its deficiency leads to disturbed thymic architecture, aberrant T cell development, and limited thymocyte expansion.For optimal visualization of CCR7 expression on different cell types it is necessary to use multi-color staining to discriminate different cell subsets as well as following the protocol. To address specificity, the staining profile of 4B12 has been compared to a polyclonal ab generated against a CCR7 peptide. This analysis confirms that the polyclonal ab and 4B12 stain similar populations of cells. Furthermore, 4B12 stains Ms CCR7-GFP fusion protein-transfected RBL cells.Applications Reported: This 4B12 ab has been reported for use in FC analysis.Applications Tested: This 4B12 ab has been tested by FC analysis of Ms splenocytes. This can be used at less than or equal to 1ug per test. A test is defined as the amount (ug) of ab that will stain a cell sample in a final volume of 100uL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. It is recommended that the ab be carefully titrated for optimal performance in the assay of interest.Important: Staining with the 4B12 monoclonal ab 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 2-8°C, results in brighter 4B12 staining, as well as better resolution between positive and negative populations. Please see data for the PE 4B12 (cat. 12-1971) which demonstrates a comparison of staining at 2-8°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 Fc gammaIIIR/Fc gammaIIR with 5ug/million cells purified anti-Ms 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 1ug/million cells 4B12 and incubate in a 37°C waterbath or at 2-8°C (please see notes above) for 0.5 hours.3. Wash cells 1X with 3 mL of FC 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 Ms CCR7 with the 4B12 ab and the CCR7 ligand CCL19-Fc (cat. 14-1972) may be difficult due to different binding conditions required for the ab 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.APC-eFluor® 780 emits at 780 nm and is excited with the Red laser (633 nm). Please make sure that your instrument is capable of detecting this fluorochome.Light sensitivity: This tandem dye is sensitive to photo-induced oxidation. Please protect this vial and stained samples from light. Fixation: Samples can be stored in IC Fixation Buffer (cat. 00-8222) (100uL of cell sample + 100uL of IC Fixation Buffer) or 1-step Fix/Lyse Solution (cat. 00-5333) for up to 3 days in the dark at 4°C with minimal impact on brightness and FRET efficiency/compensation. Some generalizations regarding fluorophore performance after fixation can be made, but clone specific performance should be determined empirically.Excitation: 633-647nm; Emission: 780nm; Laser: Red Laser.Filtration: 0.2μm post-manufacturing filtered.
|PBS with 0.09% sodium azide; pH 7.2|
|Bukitt's lymphoma receptor 2, C-C chemokine receptor type 7, CC chemokine receptor 7, CCR-7, CD197, Chemokine (C-C Motif) Receptor 7, chemokine (C-C) receptor 7, CMKBR7, EBI1, EBV-induced G protein-coupled receptor 1, EBV-induced G-protein coupled receptor 1, Epstein-Barr virus induced gene 1, Epstein-Barr Virus-Induced G-Protein Coupled Receptor 1, lymphocyte-specific G protein-coupled peptide receptor, MIP-3 Beta Receptor|
|Store at 2-8°C. Do not freeze. Light-sensitive material. This tandem dye is sensitive to photo-induced oxidation. Protect this vial from light during storage, handling and experimental procedures.|
For Research Use Only.