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H Endogenous Rabbit IgG

Flow Cytometry

Flow cytometric analysis of human peripheral blood mononuclear cells, untreated (left) or treated with anti-human CD3 (10 μg/ml, 72 hours, 37ºC) and anti-human CD28 (5 μg/ml, 72 hours, 37ºC) using CD71 (D7G9X) XP® Rabbit mAb (PE Conjugate) and co-stained with a CD3 antibody.

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Flow Cytometry Saponin Protocol

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. 0.1% Saponin in 1X PBS.
  4. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
  5. Recommended Anti-Rabbit secondary antibodies:

B. Fixation

  1. Collect cells by centrifugation and aspirate supernatant.
  2. Resuspend cells in 0.5–1 ml 1X PBS. Add appropriate volume of 16% Formaldehyde to obtain a final concentration of 2% (e.g. 875 μl PBS + 125 μl 16% Formaldehyde).
  3. Fix for 10 min in a 37°C water bath.
  4. Chill samples on ice for 1 min.

C. Permeabilization

  1. Wash cells by centrifugation and resuspend in 1 ml 0.1% Saponin/1X PBS.
  2. Incubate 30 min at room temperature.
  3. Add 2 ml of 0.1% Saponin/1X PBS and wash by centrifugation.

D. Immunostaining

  1. Resuspend cells in 100 µl of primary antibody diluted in 0.1% Saponin/1X PBS. See individual antibody datasheet or product webpage for the appropriate antibody dilution.
  2. Incubate for 1 hr at room temperature.
  3. Wash by centrifugation in 2 ml 0.1% Saponin/1X PBS.
  4. Resuspend cells in fluorochrome-conjugated secondary antibody, diluted in 0.1% Saponin/1X PBS at the recommended dilution.
  5. Incubate for 30 min at room temperature.
  6. Wash by centrifugation in 2 ml 0.1% Saponin/1X PBS.
  7. Resuspend cells in 0.5 ml 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA dye (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 30 min at room temperature.
  3. Analyze cells in DNA staining solution on flow cytometer.

posted March 2015

Protocol Id: 704

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 antibodies. Protect from light. Do not freeze.

Specificity / Sensitivity

CD71 (D7G9X) XP® Rabbit mAb (PE Conjugate) recognizes endogenous levels of total CD71 protein.

Species Reactivity: Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu146 of human CD71 protein.

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 CD71 (D7G9X) XP® Rabbit mAb #13113.

Transferrin receptor 1 (CD71, TFRC) is a type II transmembrane receptor and carrier protein responsible for the uptake of cellular iron through receptor-mediated endocytosis (1). Neutral pH at the cell surface promotes binding of the iron-binding glycoprotein transferrin (Tf) to the CD71 receptor. The receptor-ligand complex enters the cell through receptor-mediated endocytosis and is internalized into an endosome. Relatively lower endosomal pH leads to a change in the local charge environment surrounding the iron-transferrin binding site and results in the release of iron (2). The receptor-ligand complex is recycled to the cell surface where transferrin dissociates from the CD71 receptor (2). Ubiquitously expressed transferrin receptor is continuously recycled and undergoes clathrin-mediated endocytosis regardless of ligand binding state. The interaction between receptor and ligand has been studied in detail. The helical domain of CD71 directly interacts with the transferrin C-lobe and induces a conformation change in Tf to facilitate the transport process (3). Interaction between the receptor CD71 and transferrin is mediated by the membrane protein hemochromatosis (HFE). HFE binds the α-helical domain of CD71, blocking formation of the CD71-transferrin complex and inhibiting iron uptake (4,5). In addition to binding transferrin, CD71 also interacts with H-ferritin at the cell surface and transports this intracellular iron storage protein to cellular endosomes and lysosomes (6). Additional studies indicate that the transferrin receptor is an evolutionarily conserved receptor for a number or arenaviruses and at least one retrovirus (7,8). Aberrant expression of CD71 is seen in a number of cancers, including thyroid carcinomas, lymphomas, and T-lineage leukemias, suggesting a possible therapeutic role for targeted inhibition of the transferrin receptor (9,10).

1.  Ponka, P. and Lok, C.N. (1999) Int J Biochem Cell Biol 31, 1111-37.

2.  Bali, P.K. et al. (1991) Biochemistry 30, 324-8.

3.  Cheng, Y. et al. (2004) Cell 116, 565-76.

4.  Bennett, M.J. et al. (2000) Nature 403, 46-53.

5.  Feder, J.N. et al. (1998) Proc Natl Acad Sci U S A 95, 1472-7.

6.  Li, L. et al. (2010) Proc Natl Acad Sci U S A 107, 3505-10.

7.  Radoshitzky, S.R. et al. (2007) Nature 446, 92-6.

8.  Coffin, J.M. (2013) PLoS Biol 11, e1001574.

9.  Magro, G. et al. (2011) Thyroid 21, 267-77.

10.  Rodríguez, J.A. et al. (2011) Leuk Lymphoma 52, 2169-78.

Entrez-Gene Id 7037
Swiss-Prot Acc. P02786

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.

CD71 (D7G9X) XP® Rabbit mAb (PE Conjugate)