Flow cytometric analysis of human whole blood using Arginase-1 (D4E3M™) XP® Rabbit mAb (PE Conjugate) (solid line) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (dashed line). Analysis was performed on cells in the granulocyte gate.
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This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometric analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated Arginase-1 (D4E3M™) XP® Rabbit mAb #93668.
Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.
All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Methanol) #13593, or individually using the catalog numbers listed below.
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 for a full listing of cellular dyes validated for use in flow cytometry.
NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.
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.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.
NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or methanol. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. However, note that some fluorophores (including PE and APC) are damaged by methanol and thus should not be added prior to permeabilization. Conduct a small-scale experiment if you are unsure.
NOTE: Count cells using a hemocytometer or alternative method.
posted July 2009
revised June 2020
Protocol Id: 407
Arginase-1 (D4E3M™) XP® Rabbit mAb (PE Conjugate) recognizes endogenous levels of total arginase-1 protein. This antibody does not cross-react with arginase-2.
Human, Mouse, Rat
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Val47 of human arginase-1 protein.
L-arginine plays a critical role in regulating the immune system (1-3). In inflammation, cancer and certain other pathological conditions, myeloid cell differentiation is inhibited leading to a heterogeneous population of immature myeloid cells, known as myeloid-derived suppressor cells (MDSCs). MDSCs are recruited to sites of cancer-associated inflammation and express high levels of arginase-1 (4). Arginase-1 catalyzes the final step of the urea cycle converting L-arginine to L-ornithine and urea (5). Thus MDSCs increase the catabolism of L-arginine resulting in L-arginine depletion in the inflammatory microenvironment of cancer (4,6). The reduced availability of L-arginine suppresses T-cell proliferation and function and thus contributes to tumor progression (4,6). Arginase-1 is of great interest to researchers looking for a therapeutic target to inhibit the function of MDSCs in the context of cancer immunotherapy (7). In addition, research studies have demonstrated that Arginase-1 distinguishes primary hepatocellular carcinoma (HCC) from metastatic tumors in the liver, indicating its value as a potential biomarker in the diagnosis of HCC (8,9).
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