Figure 1. Treatment of NIH/3T3 cells with IFN-γ induces expression of PD-L1. The relationship between lysate protein concentration from untreated and IFN-γ treated NIH/3T3 cells and the absorbance at 450 nm as detected by FastScan™ Total PD-L1 ELISA Kit (Mouse Preferred) #41590 is shown in the upper figure. The corresponding western blot using a PD-L1 antibody (Mouse Preferred) is shown in the lower figure. After serum starvation, NIH/3T3 cells were treated with IFN-γ (25 ng/ml for 16 hr at 37ºC) and then lysed.
Figure 2. Detection of PD-L1 using FastScan™ Total PD-L1 ELISA Kit (Mouse Preferred) #41590 on a number of cell lines are shown in the upper figure. Mouse cell lines include RAW 264.7, untreated and treated with LPS, BA/F3, and KARPAS-299. KARPAS-299, a human cell line known to express PD-L1 shows no reactivity with FastScan™ Total PD-L1 ELISA Kit (Mouse Preferred) #41590. Corresponding western blot using a PD-L1 antibody (Mouse Preferred) is shown in the lower figure. KARPAS cell line source: Dr. Abraham Karpas at the University of Cambridge.
|Product Includes||Volume (with Count)||Solution Color|
|FastScan™ ELISA Microwell Strip Plate, 96 Well 53257||1 x 96 tests|
|PD-L1 Rabbit Capture mAb||1 x 1 ea||Green (Lyophilized)|
|PD-L1 Rabbit HRP-linked mAb||1 x 1 ea||Red (Lyophilized)|
|FastScan™ ELISA Capture Antibody Diluent||1 x 3 ml||Green|
|FastScan™ ELISA HRP Antibody Diluent||1 x 3 ml|
|TMB Substrate 7004||1 x 11 ml|
|STOP Solution 7002||1 x 11 ml|
|Sealing Tape||1 x 1 ea|
|ELISA Wash Buffer (20X) 9801||1 x 25 ml|
|FastScan™ ELISA Cell Extraction Buffer (5X) 69905||1 x 10 ml|
|FastScan™ ELISA Cell Extraction Enhancer Solution (50X) 25243||1 x 1 ml|
|FastScan™ ELISA Kit #41590 Positive Control Type 2||1 x 1 ea|
NOTE: Prepare solutions with deionized/purified water or equivalent.
Prepare only as much reagent as needed on the day of the experiment.
*IMPORTANT: The provided FastScan™ ELISA Cell Extraction Enhancer Solution (50X) may precipitate when stored at 4°C. To dissolve, warm briefly at 37°C and mix gently. The FastScan™ ELISA Cell Extraction Enhancer Solution (50X) can be stored at room temperature to avoid precipitation.
NOTE: The 1X Cell Extraction Buffer contains phosphatase inhibitors. Protease inhibitors should be added to the 1X Cell Extraction Buffer immediately prior to lysing cells. Additional phosphatase inhibitors can also be added (e.g. Protease/Phosphatase Inhibitor Cocktail (100X) #5872, not supplied).
For adherent cells
For suspension cells
NOTE: Equilibrate all materials and prepared reagents to room temperature prior to running the assay.
*NOTE: Certain FastScan™ ELISA Kits may require additional washes at this step. Any requirements for additional washes will be specifically noted in the product “Description” of the kit’s datasheet.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted May 2018
revised November 2018
Protocol Id: 1704
The FastScan™ Total PD-L1 ELISA Kit (Mouse Preferred) is a sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of PD-L1. To perform the assay, sample is incubated with a capture antibody conjugated with a proprietary tag and a second detection antibody linked to HRP, forming a sandwich with PD-L1 in solution. This entire complex is immobilized to the plate via an anti-tag antibody. The wells are then washed to remove unbound material. TMB is then added. The magnitude of observed signal is proportional to the quantity of PD-L1. Antibodies in kit are custom formulations specific to kit.
The FastScan™ Total PD-L1 ELISA Kit (Mouse Preferred) detects endogenous levels of PD-L1 as shown in Figure 1. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
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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