CD44 Monoclonal Antibody (IM7), NovaFluor™ Red 700, eBioscience™

Product Specific Information

Description
The IM7 monoclonal antibody reacts with all isoforms of mouse CD44 (Pgp-1). CD44 is expressed by hematopoietic and non-hematopoietic cells. Bone marrow myeloid cells and memory T cells highly express this antigen and peripheral B and T cells can upregulate the expression of CD44. CD44 functions as an adhesion molecule through its binding to hyaluronate, an extracellular matrix component.

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

Applications Tested
The IM7 antibody has been tested by flow cytometric analysis of mouse bone marrow cells and splenocytes. This can be used at less than or equal to 0.6 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: 637 nm; Emission: 701 nm; Laser: 633-640 nm (Red) Laser

Target Information

CD44 cell surface antigen is a 100 kDa type 1 transmembrane glycoprotein widely expressed on human leucocytes, white matter of the brain and by some epithelial cells of the intestine and breast. Several isoforms of CD44 exist, including the predominant CD44H isoform detected in many normal tissues. CD44 is a receptor for hyaluronic acid (HA) and is involved in cell-cell interactions, cell adhesion and migration. CD44 also participates in a wide variety of cellular functions including lymphocyte activation, recirculation and homing. CD44 expression may be up-regulated upon some carcinomas, and it has been speculated that this may be related to metastatic potential. CD44 is expressed by hematopoietic, non-hematopoietic cells, epithelial tissues, and to filopodia in cultured keratinocytes. Further, bone marrow myeloid cells and memory T cells express CD44 at high levels, and peripheral B and T cells can upregulate the expression of CD44 in response to certain stimulatory events. Transcripts for the CD44 gene undergo complex alternative splicing that results in many functionally distinct isoforms, however, the full-length nature of some of these variants have not been determined. Alternative splicing is the basis for the structural and functional diversity of the CD44 protein. Diseases associated with CD44 dysfunction include superficial keratitis and lichen sclerosus. CD44 also may be related to tumor metastasis formation.