CD45.1 Monoclonal Antibody (A20), NovaFluor™ Blue 660-40S, eBioscience™

Product Specific Information

Description
The A20 monoclonal antibody reacts with the mouse CD45 molecule, the leukocyte common antigen (LCA) in CD45.1-expressing mouse strains. The strains that express CD45.1 include SJL/J, DA, STS/A and RIII. CD45.1 is expressed by all leukocytes in these strains.

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

Applications Tested
The A20 antibody has been tested by flow cytometric analysis of mouse splenocytes. This can be used at less than or equal to 0.5 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: 492 nm; Emission: 665 nm; Laser: 488 nm (Blue) Laser

Target Information

CD45 (LCA, leukocyte common antigen) is a receptor-type protein tyrosine phosphatase ubiquitously expressed in all nucleated hematopoietic cells, comprising approximately 10% of all surface proteins in lymphocytes. CD45 glycoprotein is crucial in lymphocyte development and antigen signaling, serving as an important regulator of Src-family kinases. CD45 protein exists as multiple isoforms as a result of alternative splicing; these isoforms differ in their extracellular domains, whereas they share identical transmembrane and cytoplasmic domains. These CD45 isoforms differ in their ability to translocate into the glycosphingolipid-enriched membrane domains and their expression depends on cell type and physiological state of the cell. Besides the role in immunoreceptor signaling, CD45 is important in promoting cell survival by modulating integrin-mediated signal transduction pathway and is also involved in DNA fragmentation during apoptosis. CD45RA is an isoform of the CD45 complex and has restricted expression between different subtypes of lymphoid cells.