Confocal immunofluorescent analysis of HCC1954 (left), MDA-MB-231 (center), and A549 (right) cells using PD-L1 (Extracellular Domain Specific) (D8T4X) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Flow cytometric analysis of Ramos cells (blue) and MDA-MB-231 cells (green) using PD-L1 (Extracellular Domain Specific) (D8T4X) Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:
NOTE: Cells should be grown, treated, fixed and stained directly in multiwell plates, chamber slides or on coverslips.
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
posted November 2006
revised December 2010
Protocol Id: 32
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com/flowdyes for a listing of cellular dyes validated for use in flow cytometry.
NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.
NOTE: If live cell staining is desired, proceed to Section C.
NOTE: Count cells using a hemocytometer or alternative method.
posted January 2009
revised November 2019
Protocol Id: 133
PD-L1 (Extracellular Domain Specific) (D8T4X) Rabbit mAb recognizes endogenous levels of total PD-L1 protein.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with mammalian cells expressing full length PD-L1 protein.
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).
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Alexa Fluor is a registered trademark of Life Technologies Corporation.
DRAQ5 is a registered trademark of Biostatus Limited.