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PD-L1 (Extracellular Domain Specific) (D8T4X) Rabbit mAb (Pacific Blue™ Conjugate) #55919
Gallery: PD-L1 (Extracellular Domain Specific) (D8T4X) Rabbit mAb (Pacific Blue™ Conjugate) #55919
Flow Cytometry, Extracellular Epitope Protocol for Direct Conjugates
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
- 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
- Formaldehyde (methanol free).
- Incubation Buffer: Dissolve 0.5 g bovine serum albumin (BSA) (#9998) in 100mL 1X PBS. Store at 4°C.
NOTE: If live cell staining is desired, proceed to Section C.
- Collect cells by centrifugation and aspirate supernatant.
- Resuspend cells in 0.5-1 ml PBS. Add formaldehyde to obtain a final concentration of 4% formaldehyde.
- Fix for 15 minutes at room temperature.
- Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 1X PBS.
- Proceed with staining or store cells at +4°C in PBS with 0.1% sodium azide.
NOTE: Count cells using a hemocytometer or alternative method.
- Aliquot desired number of cells into tubes or wells.
- If necessary, centrifuge to remove excess PBS.
- Resuspend cells in 100 µl of diluted antibody conjugate (prepared in incubation buffer at the recommended dilution).
- Incubate for 30-60 minutes at room temperature.
- Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
- Resuspend cells in 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to Section D.
D. Optional DNA Dye
- Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
- Incubate for at least 5 min at room temperature.
- Analyze cells in DNA staining solution on flow cytometer.
posted January 2009
revised June 2017
PD-L1 (Extracellular Domain Specific) (D8T4X) Rabbit mAb (Pacific Blue™ Conjugate) recognizes endogenous levels of total PD-L1 protein.Species Reactivity: Human
Monoclonal antibody is produced by immunizing animals with mammalian cells expressing full length PD-L1 protein.
This Cell Signaling Technology antibody is conjugated to Pacific Blue™ fluorescent dye and tested in-house for direct flow cytometry in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated antibody PD-L1 (Extracellular Domain Specific) (D8T4X) Rabbit mAb #86744.
Programmed cell death 1 ligand 1 (PD-L1, B7-H1, CD274) is a member of the B7 family of cell surface ligands that regulate T cell activation and immune responses. The PD-L1 ligand binds the PD-1 transmembrane receptor and inhibits T cell activation. PD-L1 was discovered following a search for novel B7 protein homologs and was later shown to be expressed by antigen presenting cells, activated T cells, and tissues including placenta, heart, and lung (1-3). Similar in structure to related B7 family members, PD-L1 protein contains extracellular IgV and IgC domains and a short, cytoplasmic region. Research studies demonstrate that PD-L1 is expressed in several tumor types, including melanoma, ovary, colon, lung, breast, and renal cell carcinomas (4-6). Expression of PD-L1 in cancer is associated with tumor infiltrating lymphocytes, which mediate PD-L1 expression through the release of interferon gamma (7). Additional research links PD-L1 expression to cancers associated with viral infections (8,9).
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. Pacific Blue is a trademark of Molecular Probes, Inc.