Upstream / Downstream

pathwayImage

Explore pathways related to this product.

Friends and Family

25% Off

the purchase of 3 or more products

Shop

To Purchase # 92734S

92734S 100 µl (50 tests) $329.00.0
$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 Mk Endogenous Rabbit IgG
Image
Image
Image
Image

Flow Cytometry

Flow cytometric analysis of human whole blood cells stained with VISTA (D1L2G™) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) and co-stained with CD19-PE and CD14-FITC. VISTA is present on CD14+ Monocytes (bottom right; upper right quadrant) and shows an increase in intensity when compared to the isotype control (bottom left; upper left quadrant). CD19+ B cells are negative for VISTA (top right; upper left quadrant) and do not show an increase in intensity when compared to the isotype control (top left; upper left quadrant).

Learn more about how we get our images
Image
Image
Page

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

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

VISTA (D1L2G™) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) recognizes endogenous levels of total VISTA protein.


Species Reactivity: Human, Monkey

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human VISTA protein.

Product Description

This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 647 fluorescent dye 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 VISTA (D1L2G™) XP® Rabbit mAb #64953.


VISTA (V-Domain Ig Suppressor of T Cell Activation) is a negative checkpoint control protein that regulates T cell activation and immune responses. VISTA, which contains a single Ig-like V-type domain, a transmembrane domain, and an intracellular domain, has sequence similarity to both the B7 and CD28 family members. Although primarily expressed by myeloid cells, VISTA is also expressed by CD4+, CD8+, and FoxP3+ T-cells. Thus, VISTA is described as both a ligand and a receptor (1-3). Blocking VISTA induces T-cell activation and proliferation, and potentiates disease severity in the EAE model (1). Furthermore, genetic deletion of VISTA in mice leads to spontaneous T-cell activation and chronic inflammation (4,5). In mouse models of cancer, neutralization of VISTA enhances T-cell proliferation and effector function and increases tumor infiltration, suggesting VISTA blockade could be an effective strategy for tumor immunotherapy (6,7).


1.  Wang, L. et al. (2011) J Exp Med 208, 577-92.

2.  Flies, D.B. et al. (2011) J Immunol 187, 1537-41.

3.  Lines, J.L. et al. (2014) Cancer Res 74, 1924-32.

4.  Wang, L. et al. (2014) Proc Natl Acad Sci U S A 111, 14846-51.

5.  Liu, J. et al. (2015) Proc Natl Acad Sci U S A 112, 6682-7.

6.  Le Mercier, I. et al. (2014) Cancer Res 74, 1933-44.

7.  Lines, J.L. et al. (2014) Cancer Immunol Res 2, 510-7.


Entrez-Gene Id 64115
Swiss-Prot Acc. Q9H7M9


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
D1L2G is a trademark of Cell Signaling Technology. Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
The Alexa Fluor dye conjugates in this product are sold under license from Life Technologies Corporation, for research use only excluding use in combination with DNA microarrays and high content screening (HCS).
This product is provided under an intellectual property license from Life Technologies Corporation. The transfer of this product is contingent on the buyer using the purchased product solely in research, including use with HCS or other automated imaging applications but excluding use in combination with DNA microarrays. The buyer must not sell or otherwise transfer this product or its components for (a) diagnostic, therapeutic or prophylactic purposes; (b) testing, analysis or screening services, or information in return for compensation on a per-test basis; (c) manufacturing or quality assurance or quality control, or (d) resale, whether or not resold for use in research. For information on purchasing a license to this product for purposes other than as described above, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or outlicensing@lifetech.com.

92734
VISTA (D1L2G™) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate)