CD73 Monoclonal Antibody (eBioTY/11.8 (TY/11.8)), NovaFluor™ Red 755, eBioscience™

Product Specific Information

Description
eBioTY/11.8 recognizes the with CD73 a 69-kDa GPI-anchored cell-surface protein with ecto-5'-nucleotidase activity. Expression on myeloid cells (CD11b) is restricted to the bone marrow. In human CD73 can be induced to secrete a soluble form with IL-2 suggesting a role in mediating activation signals. Differences between human and mouse CD73 have been reported. BALB/c mice have more CD4+CD73+ than CD8+CD73+ while the reciprocal is documented in humans.

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

Applications Tested
This eBioTY/11.8 (TY/11.8) antibody has been tested by flow cytometric analysis of mouse splenocytes. This can be used at less than or equal to 0.2 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

Excitation: 636 nm; Emission: 755 nm; Laser: 633-640 nm (Red) Laser

Target Information

CD73, also known as Ecto-5-prime-nucleotidase or 5-prime-ribonucleotide phosphohydrolase, is an enzyme that catalyzes the conversion of purine 5-prime mononucleotides to nucleosides at neutral pH, with AMP as its preferred substrate. It is composed of a dimer of two identical 70 kDa subunits, externally bound to the plasma membrane via a glycosyl phosphatidyl inositol linkage. CD73 serves as a marker of lymphocyte differentiation and is expressed on a subset of lymphocytes, increasing during lymphocyte maturation. It is found on memory CD4 T cells, which resemble uncommitted primed precursor helper cells (Thpp) capable of differentiating into Th1 or Th2 cells, and is also present on regulatory T cells. CD73 deficiency is associated with various immunodeficiency diseases. Other forms of 5-prime nucleotidase exist in the cytoplasm and lysosomes, distinguishable from CD73 by their substrate affinities, requirement for divalent magnesium ions, activation by ATP, and inhibition by inorganic phosphate. The CD73 gene is localized to chromosome 6q14-q21, and defects in this gene can lead to conditions such as calcification of joints and arteries, and intestinal tuberculosis. Additionally, two transcript variants encoding different isoforms of CD73 have been identified.