CD274 (PD-L1, B7-H1) Monoclonal Antibody (MIH5), NovaFluor™ Yellow 690, eBioscience™

Product Specific Information

Description
The MIH5 monoclonal antibody reacts with mouse B7-H1, also known as PD-L1. B7-H1, a member of the B7 family, has a predicted molecular weight of approximately 40 kDa and belongs to the Ig superfamily. B7-H1 is expressed on a majority of leukocytes including T, B, NK and DC. B7-H1 is a ligand for PD-1. Interaction of PD-1 with either PD-L1 (B7-H1) or PD-L2 (B7-DC) results in inhibition of T and B cell responses. MIH5 is reported to be a blocking antibody.

This product contains 1 vial of NovaFluor conjugate and 1 vial of CellBlox Plus Blocking Buffer.

Applications Tested
The MIH5 antibody has been tested by flow cytometric analysis of mouse splenocytes. This can be used at less than or equal to 0.06 µg per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest.

Master mixes
• Master mixes of NFs should be made at 2-8 °C and may be made up to 4 hours ahead of time.
• We do not recommend storing master mixes containing NovaFluor conjugates overnight or longer.

Whole Blood compatibility
• When utilizing whole blood (as opposed to density-gradient-purified PBMC), we recommend lysing red blood cells in bulk prior to staining with NovaFluor conjugates.
• See the Bulk Lysis of Human Whole Blood protocol here.
• Staining of whole blood with NovaFluor conjugates followed by lysis of red blood cells may result in higher-than-expected background staining.

Viability dye compatibility
• NovaFluor dyes are not compatible with DNA intercalating viability dyes.
• Do not use viability dyes such as propidium iodide, 7-actinomycin D (7-AAD) and DAPI. Invitrogen LIVE/DEAD Fixable Dead Cell stains are recommended for use with NovaFluor dyes.

CellBlox Plus Blocking Buffer
• This NovaFluor conjugate comes with CellBlox Plus Blocking Buffer (for 25 µg or 25 test products, Cat. No. C001T02F01; for 100 µg or 100 test, Cat. No. C001T03F01), essential for optimal staining.
• Use CellBlox Plus Blocking Buffer in all experiments with NovaFluor conjugates.
• Add 5 µL per sample to antibody cocktails/master mixes (regardless of how many Novafluor-conjugated antibodies are present) before combining with cells.
• CellBlox Plus Blocking Buffer is compatible with either Super Bright Complete Blocking Buffer or Brilliant Stain Buffer and can be used in antibody cocktails/master mixes with those reagents.
• For single-color controls, use 5 µL of CellBlox Plus Blocking Buffer per 100 µL of cell sample (10^3 to 10^8 cells).

NovaFluor conjugates are based on Phiton technology utilizing novel fluorophore-containing nucleic acid dye structures that allow for engineered fluorescent signatures with consideration for spillover and spread impacts. Learn more

Our internal testing shows that NovaFluor Yellow 690 non-specifically stains B cells in SJL mice. Non-specific staining has not been observed in BALB/c or C57BL/6 mice. Other strains have not been tested. See the Antibody Testing Data for an example of this strain-dependent difference.

Excitation: 551 nm; Emission: 691 nm; Laser: 561 nm (Yellow) Laser

Target Information

Programmed death receptor ligand 1 (PD-L1, also called B7-H1) is a recently described B7 family member. To date, one specific receptor has been identified that can be ligated by PD-L1. This receptor, programmed death receptor 1 (PD-1), has been shown to negatively regulate T-cell receptor (TCR) signaling. Upon ligating its receptor, PD-L1 has been reported to decrease TCR-mediated proliferation and cytokine production. PD-L1 expression was found to be abundant on many murine and human cancers and could be further up-regulated upon IFN-gamma stimulation. Thus, PD-L1 might play an important role in tumor immune evasion.