Flow cytometric analysis of live human peripheral blood mononuclear cells using CD45 (HI30) Mouse mAb (violetFluor™ 450 Conjugate) (solid line) compared to concentration-matched Mouse (MOPC-21) mAb IgG1 Isotype Control (violetFluor™ 450 Conjugate) #40282 (dashed line).
This Cell Signaling Technology antibody is conjugated to violetFluor™ 450 and tested in-house for direct flow cytometric analysis in human cells.
Supplied in 10 mM NaH2PO4, 150 mM NaCl, 0.09% NaN3, 0.1% gelatin, pH7.2. Do not aliquot the antibody. Protect from light. Do not freeze.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com/flowdyes for a listing of cellular dyes validated for use in flow cytometry.
NOTE: Count cells using a hemocytometer or alternative method.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to Immunostaining.
NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.
posted June 2017
revised August 2019
Protocol Id: 1504
CD45 (HI30) Mouse mAb (violetFluor™ 450 Conjugate) recognizes endogenous levels of total CD45 protein. This antibody detects an epitope within the extracellular domain.Species Reactivity:
This monoclonal antibody was purified from tissue culture supernatant via affinity chromatography. The purified antibody was conjugated under optimal conditions, with unreacted dye removed from the preparation.
The protein phosphatase (PTP) receptor CD45 is a type I transmembrane protein comprised of a pair of intracellular tyrosine phosphatase domains and a variable extracellular domain generated by alternative splicing (1). The catalytic activity of CD45 is a function of the first phosphatase domain (D1) while the second phosphatase domain (D2) may interact with and stabilize the first domain, or recruit/bind substrates (2,3). CD45 interacts directly with antigen receptor complex proteins or activates Src family kinases involved in the regulation of T- and B-cell antigen receptor signaling (1). Specifically, CD45 dephosphorylates Src-family kinases Lck and Fyn at their conserved negative regulatory carboxy-terminal tyrosine residues and upregulates kinase activity. Conversely, studies indicate that CD45 can also inhibit Lck and Fyn by dephosphorylating their positive regulatory autophosphorylation site. CD45 appears to be both a positive and a negative regulator that conducts signals depending on specific stimuli and cell type (1). Human leukocytes including lymphocytes, eosinophils, monocytes, basophils, and neutrophils express CD45, while erythrocytes and platelets are negative for CD45 expression (4).
The HI30 antibody is widely used as a marker for human CD45 expression on all leukocytes, including T cells, B cells, monocytes, macrophages, and NK cells.
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Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
violetFluor is a registered trademark of Tonbo Biosciences.