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TRAIL (C92B9) Rabbit mAb (PE Conjugate)
Antibody Conjugates

TRAIL (C92B9) Rabbit mAb (PE Conjugate) #37020


H Endogenous Rabbit IgG
Flow Cytometry

Flow cytometric analysis of 786-0 cells using TRAIL (C92B9) Rabbit mAb (PE Conjugate) (blue) compared to a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (red).

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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

Application Dilutions
Flow Cytometry 1:50

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.

TRAIL (C92B9) Rabbit mAb (PE Conjugate) detects endogenous levels of total human TRAIL protein.

Species Reactivity:


Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Lys60 of human TRAIL, within the extracellular region of the protein.

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 TRAIL (C92B9) Rabbit mAb #3219.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), also referred to as Apo2 ligand, first identified based on its sequence homology to TNF and Fas/Apo ligand is a member of the TNF family of cytokines and either exists as a type II membrane or soluble protein (1,2). TRAIL induces apoptosis in a variety of transformed cell lines and plays a role in anti-tumor and anti-viral immune surveillance (3). TRAIL signals via binding with death receptors DR4 (TRAIL-R1) (4) and DR5 (TRAIL-R2) (5-8) which can trigger apoptosis as well as NF-κB activation (7,9). Death domains on these receptors leads to the recruitment of a death-induced signaling complex (DISC) leading to caspase-8 and subsequent caspase-3 activation. In addition, TRAIL binds with decoy receptors DcR1 (TRAIL-R3) (10-13) and DcR2 (TRAIL-R4, TRUNDD) (14-15) which lack the functional cytoplasmic death domain antagonizing TRAIL-induced apoptosis. Osteoprotegerin (OPG) has also been identified as receptor capable of inhibiting TRAIL-induced apoptosis (16). The selectivity of soluble TRAIL at triggering apoptosis in transformed cells as compared to normal cells has led to its investigation as a potential cancer therapeutic (17-18).

  1. Wiley, S.R. et al. (1995) Immunity 3, 673-82.
  2. Pitti, R.M. et al. (1996) J Biol Chem 271, 12687-90.
  3. Almasan, A. and Ashkenazi, A. Cytokine Growth Factor Rev 14, 337-48.
  4. Pan, G. et al. (1997) Science 276, 111-3.
  5. Walczak, H. et al. (1997) EMBO J 16, 5386-97.
  6. MacFarlane, M. et al. (1997) J Biol Chem 272, 25417-20.
  7. Chaudhary, P.M. et al. (1997) Immunity 7, 821-30.
  8. Schneider, P. et al. (1997) FEBS Lett 416, 329-34.
  9. Shetty, S. et al. (2002) Apoptosis 7, 413-20.
  10. Sheridan, J.P. et al. (1997) Science 277, 818-21.
  11. Degli-Esposti, M.A. et al. (1997) J Exp Med 186, 1165-70.
  12. MacFarlane, M. et al. (1997) J Biol Chem 272, 25417-20.
  13. Schneider, P. et al. (1997) FEBS Lett 416, 329-34.
  14. Pan, G. et al. (1998) FEBS Lett 424, 41-5.
  15. Marsters, S.A. et al. (1997) Curr Biol 7, 1003-6.
  16. Kelley, S.K. et al. (2001) J Pharmacol Exp Ther 299, 31-8.
  17. Walczak, H. et al. (1999) Nat Med 5, 157-63.
  18. Ashkenazi, A. et al. (1999) J Clin Invest 104, 155-62.
Entrez-Gene Id
Swiss-Prot Acc.
For Research Use Only. Not For Use In Diagnostic Procedures.

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

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100 µl  (50 tests) $ 305.0

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