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65396
M-CSF Receptor (D3O9X) XP® Rabbit mAb (PE Conjugate)
Antibody Conjugates

M-CSF Receptor (D3O9X) XP® Rabbit mAb (PE Conjugate) #65396

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Flow cytometric analysis of human peripheral blood mononuclear cells co-stained with anti-human CD14-APC using M-CSF Receptor (D3O9X) XP® Rabbit mAb (PE Conjugate) (right) compared to a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (left).

To Purchase # 65396S
Product # Size Price
65396S
100 µl  (50 tests) $ 348

Supporting Data

REACTIVITY H
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 cytometry analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated M-CSF Receptor (D3O9X) XP® Rabbit mAb #67455.

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, Extracellular Epitope 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. Formaldehyde (methanol free).
  3. Incubation Buffer: Dissolve 0.5 g bovine serum albumin (BSA) (#9998) in 100mL 1X PBS. Store at 4°C.

B. Fixation

NOTE: If live cell staining is desired, proceed to Section C.

  1. Collect cells by centrifugation and aspirate supernatant.
  2. Resuspend cells in 0.5-1 ml PBS. Add formaldehyde to obtain a final concentration of 4% formaldehyde.
  3. Fix for 15 minutes at room temperature.
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 1X PBS.
  5. Proceed with staining or store cells at +4°C in PBS with 0.1% sodium azide.

C. Immunostaining

NOTE: Count cells using a hemocytometer or alternative method.

  1. Aliquot desired number of cells into tubes or wells.
  2. If necessary, centrifuge to remove excess PBS.
  3. Resuspend cells in 100 µl of diluted antibody conjugate (prepared in incubation buffer at the recommended dilution).
  4. Incubate for 30-60 minutes at room temperature.
  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 Section D.

D. 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 January 2009

revised June 2017

Protocol Id: 180

Specificity / Sensitivity

M-CSF Receptor (D3O9X) XP® Rabbit mAb (PE Conjugate) recognizes endogenous levels of total M-CSF Receptor protein.

Species Reactivity:

Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with recombinant M-CSF Receptor protein.

Background

Macrophage-colony stimulating factor (M-CSF, CSF-1) receptor is an integral membrane tyrosine kinase encoded by the c-fms proto-oncogene. M-CSF receptor is expressed in monocytes (macrophages and their progenitors) and drives growth and development of this blood cell lineage. (1-3). Binding of M-CSF to its receptor induces receptor dimerization, activation, and autophosphorylation of cytoplasmic tyrosine residues used as docking sites for SH2-containing signaling proteins (4). There are at least five major tyrosine autophosphorylation sites. Tyr723 (Tyr721 in mouse) is located in the kinase insert (KI) region. Phosphorylated Tyr723 binds the p85 subunit of PI3 kinase as well as PLCγ2 (5). Phosphorylation of Tyr809 provides a docking site for Shc (5). Overactivation of this receptor can lead to a malignant phenotype in various cell systems (6). The activated M-CSF receptor has been shown to be a predictor of poor outcome in advanced epithelial ovarian carcinoma (7) and breast cancer (8).

  1. Stanley, E.R. et al. (1978) Nature 274, 168-70.
  2. Byrne, P.V. et al. (1981) J Cell Biol 91, 848-53.
  3. Bourette, R.P. and Rohrschneider, L.R. (2000) Growth Factors 17, 155-66.
  4. Novak, U. et al. (1996) Oncogene 13, 2607-13.
  5. Bourette, R.P. et al. (1997) EMBO J 16, 5880-93.
  6. Morley, G.M. et al. (1999) Oncogene 18, 3076-84.
  7. Toy, E.P. et al. (2001) Gynecol Oncol 80, 194-200.
  8. Maher, M.G. et al. (1998) Clin Cancer Res 4, 1851-6.

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.