CD54 (ICAM-1) Monoclonal Antibody (YN1/1.7.4), NovaFluor™ Yellow 700, eBioscience™

Product Specific Information

Description
The YN1/1.7.4 monoclonal antibody reacts with mouse CD54 (InterCellular Adhesion Molecule-1, ICAM-1), a 90-110 kDa transmembrane glycoprotein expressed by monocytes, lymphocytes, dendritic cells, and endothelial cells. Expression of CD54 is upregulated on activated lymphocytes. Interaction of CD54 with its ligands CD11a and CD11b is important in the inflammatory response. The YN1/1.7.4 monoclonal antibody recognizes a different epitope than the eBioKAT-1 monclonal antibody.

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

Applications Tested
The YN1/1.7.4 antibody has been tested by flow cytometric analysis of mouse splenocytes. This can be used at less than or equal to 0.4 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 700 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: 701 nm; Laser: 561 nm (Yellow) Laser

Target Information

ICAM-1 (CD54) is an 85-110 kDa single-chain type 1 integral membrane glycoprotein with an extracellular domain of five immunoglobulin superfamily repeats, a transmembrane region and a cytoplasmic domain. ICAM-1 has 7 potential N-linked glycosylation sites and shares considerable amino acid sequence homology with ICAM-3 (CD50) and ICAM-2 (CD102). ICAM-1 binds to integrins of type CD11a/CD18 (leukocyte adhesion molecule, LFA-1), or CD11b/CD18 (Mac-1) and is exploited by Rhinovirus as a receptor. ICAM-1 is expressed by activated endothelial cells and detected on epithelial cells, fibroblasts, chondrocytes, B lymphocytes, T lymphocytes (low), monocytes, macrophages, dendritic cells and neutrophils, with lower levels that increase upon inflammation. ICAM-1 is also detected in some carcinoma and melanoma cells. Soluble ICAM-1 is detectable in the plasma and is elevated in patients with various inflammatory syndromes.