CD63 Monoclonal Antibody (H5C6), NovaFluor™ Yellow 700, eBioscience™

Product Specific Information

Description
This H5C6 monoclonal antibody reacts with human CD63, a type III member of the tetraspanin family of transmembrane proteins.

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

Applications Tested
This H5C6 antibody has been pre-titrated and tested by flow cytometric of normal human peripheral blood cells. This can be used at 5 µL (1.0 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.

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: 551 nm; Emission: 701 nm; Laser: 561 nm (Yellow) Laser

Target Information

CD63, also known as LAMP-3, is a glycoprotein belonging to the tetraspanin family, primarily located in late endosomes, lysosomes, and secretory vesicles across various cell types. It is expressed intracellularly in resting platelets and basophils, but upon activation, CD63 translocates to the plasma membrane, making it a widely used marker for basophil activation. Unlike basophils, mast cells can expose CD63 without activation. CD63 is expressed on activated platelets, monocytes, macrophages, and granulocytes, as well as endothelial cells, fibroblasts, and smooth muscle cells. It interacts with integrins (such as VLA-3 and VLA-6) and TIMP-1, playing roles in mediating allergic responses, phagocytosis, and cell migration. Additionally, CD63 is involved in the regulation of H/K-ATPase trafficking and ROMK1 channels, and serves as a T-cell costimulation molecule. CD63 expression can be used to predict prognosis in early-stage carcinomas and is identical to the melanoma-associated antigen ME491 and the platelet antigen PTLGP40. Diseases associated with CD63 dysfunction include melanoma and Hermansky-Pudlak Syndrome, highlighting its significance in both immune responses and disease processes.