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86352
Arginase-1 (D4E3M™) XP® Rabbit mAb (PE Conjugate)
Antibody Conjugates

Arginase-1 (D4E3M™) XP® Rabbit mAb (PE Conjugate) #86352

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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.

To Purchase # 86352S
Product # Size Price
86352S
100 µl  (50 tests) N/A

Supporting Data

REACTIVITY H M R
SENSITIVITY Endogenous
MW (kDa)
Isotype Rabbit IgG

Product Description

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.

Product Usage Information

Application Dilutions
Flow Cytometry 1:50

Storage:

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.

Protocol

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Flow Cytometry, Methanol Permeabilization Protocol for Direct Conjugates

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 16% Formaldehyde (methanol free).
  3. 100% methanol.
  4. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

B. Fixation

NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.

  1. Collect cells by centrifugation and aspirate supernatant.
  2. Resuspend cells in 0.5-1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
  3. Fix for 15 min at room temperature.
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS.

C. Permeabilization

  1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
  2. Incubate 30 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

  1. Aliquot desired number of cells into tubes or wells.
  2. Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
  3. Resuspend cells in 100 µl of diluted antibody conjugate (prepared in incubation buffer at the recommended dilution).
  4. Incubate for 1 hr at room temperature. Protect from light.
  5. Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
  6. Resuspend cells in 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section E).

E. Optional DNA Dye

  1. Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
  2. Incubate for at least 5 min at room temperature.
  3. Analyze cells in DNA staining solution on flow cytometer.

posted July 2009

revised June 2017

Protocol Id: 407

Specificity / Sensitivity

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.

Species Reactivity:

Human, Mouse, Rat

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Val47 of human arginase-1 protein.

Background

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).

  1. Albina, J.E. et al. (1989) J Exp Med 169, 1021-9.
  2. Mills, C.D. (2001) Crit Rev Immunol 21, 399-425.
  3. Rodriguez, P.C. et al. (2004) Cancer Res 64, 5839-49.
  4. Gabrilovich, D.I. and Nagaraj, S. (2009) Nat Rev Immunol 9, 162-74.
  5. Wu, G. and Morris, S.M. (1998) Biochem J 336 ( Pt 1), 1-17.
  6. Raber, P. et al. (2012) Immunol Invest 41, 614-34.
  7. Wesolowski, R. et al. (2013) J Immunother Cancer 1, 10.
  8. Sang, W. et al. (2015) Tumour Biol 36, 3881-6.
  9. Geramizadeh, B. and Seirfar, N. (2015) Hepat Mon 15, e30336.

Pathways & Proteins

Explore pathways + proteins related to this product.

For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.

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