Upstream / Downstream

pathwayImage

Explore pathways related to this product.

Friends and Family

25% Off

the purchase of 3 or more products

Shop

To Purchase # 55919S

55919S 100 µl (50 tests) $299.00.0
$0.00

Questions?

Find answers on our FAQs page.

ANSWERS  

PhosphoSitePlus® Resource

  • Additional protein information
  • Analytical tools

LEARN MORE

REACTIVITY SENSITIVITY MW (kDa) Isotype
H Endogenous Rabbit IgG
Image
Image
Image
Image

Flow Cytometry

Flow cytometric analysis of SUP-M2 cells (green), MDA-MB-231 cells (blue), and Ramos cells (red) using using PD-L1 (Extracellular Domain Specific) (D8T4X) Rabbit mAb (Pacific Blue™ Conjugate).

Learn more about how we get our images
Image
Image
Page

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.

D. 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

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.

Specificity / Sensitivity

PD-L1 (Extracellular Domain Specific) (D8T4X) Rabbit mAb (Pacific Blue™ Conjugate) recognizes endogenous levels of total PD-L1 protein.


Species Reactivity: Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with mammalian cells expressing full length PD-L1 protein.

Product Description

This Cell Signaling Technology antibody is conjugated to Pacific Blue™ fluorescent dye and tested in-house for direct flow cytometry in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated antibody PD-L1 (Extracellular Domain Specific) (D8T4X) Rabbit mAb #86744.


Programmed cell death 1 ligand 1 (PD-L1, B7-H1, CD274) is a member of the B7 family of cell surface ligands that regulate T cell activation and immune responses. The PD-L1 ligand binds the PD-1 transmembrane receptor and inhibits T cell activation. PD-L1 was discovered following a search for novel B7 protein homologs and was later shown to be expressed by antigen presenting cells, activated T cells, and tissues including placenta, heart, and lung (1-3). Similar in structure to related B7 family members, PD-L1 protein contains extracellular IgV and IgC domains and a short, cytoplasmic region. Research studies demonstrate that PD-L1 is expressed in several tumor types, including melanoma, ovary, colon, lung, breast, and renal cell carcinomas (4-6). Expression of PD-L1 in cancer is associated with tumor infiltrating lymphocytes, which mediate PD-L1 expression through the release of interferon gamma (7). Additional research links PD-L1 expression to cancers associated with viral infections (8,9).


1.  Dong, H. et al. (1999) Nat Med 5, 1365-9.

2.  Freeman, G.J. et al. (2000) J Exp Med 192, 1027-34.

3.  Liang, S.C. et al. (2003) Eur J Immunol 33, 2706-16.

4.  Dong, H. et al. (2002) Nat Med 8, 793-800.

5.  Thompson, R.H. et al. (2006) Cancer Res 66, 3381-5.

6.  Pardoll, D.M. (2012) Nat Rev Cancer 12, 252-64.

7.  Taube, J.M. et al. (2012) Sci Transl Med 4, 127ra37.

8.  Lyford-Pike, S. et al. (2013) Cancer Res 73, 1733-41.

9.  Chen, B.J. et al. (2013) Clin Cancer Res 19, 3462-73.


Entrez-Gene Id 29126
Swiss-Prot Acc. Q9NZQ7


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Pacific Blue is a trademark of Molecular Probes, Inc.

55919
PD-L1 (Extracellular Domain Specific) (D8T4X) Rabbit mAb (Pacific Blue™ Conjugate)