Flow cytometric analysis of fixed and permeabilized human whole blood cells stained with VISTA (D1L2G™) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (Alexa Fluor® 488 Conjugate) #2975 and co-stained with CD19-APC or CD14-APC. 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).
This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 488 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.
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.
All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Methanol) #13593, or individually using the catalog numbers listed below.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com for a full listing of cellular dyes validated for use in flow cytometry.
NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.
NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.
NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or methanol. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. However, note that some fluorophores (including PE and APC) are damaged by methanol and thus should not be added prior to permeabilization. Conduct a small-scale experiment if you are unsure.
NOTE: Count cells using a hemocytometer or alternative method.
posted July 2009
revised June 2020
Protocol Id: 407
VISTA (D1L2G™) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) recognizes endogenous levels of total VISTA protein.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human VISTA protein.
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).
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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 conditioned on the buyer using the purchased product solely in research conducted by the buyer, excluding contract research or any fee for service research, and the buyer must not (1) use 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; or (c) manufacturing or quality assurance or quality control, and/or (2) sell or transfer this product or its components for 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 [email protected]