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15631
VCAM-1 (D8U5V) Rabbit mAb (Mouse Specific) (Alexa Fluor® 488 Conjugate)
Antibody Conjugates

VCAM-1 (D8U5V) Rabbit mAb (Mouse Specific) (Alexa Fluor® 488 Conjugate) #15631

APPLICATIONS

REACTIVITY SENSITIVITY MW (kDa) Isotype
M Endogenous Rabbit IgG
Flow Cytometry

Flow cytometric analysis of A20 cells (blue) and MS1 cells (green) using VCAM-1 (D8U5V) Rabbit mAb (Mouse Specific) (Alexa Fluor® 488 Conjugate) (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (Alexa Fluor® 488 Conjugate) #2975 (dashed lines).

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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
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.

VCAM-1 (D8U5V) Rabbit mAb (Mouse Specific) (Alexa Fluor® 488 Conjugate) recognizes endogenous levels of total VCAM-1 protein. This antibody recognizes both long and short isoforms of VCAM-1 protein.

Species Reactivity:

Mouse

Monoclonal antibody is produced by immunizing animals with recombinant mouse VCAM-1 protein.

This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 488 fluorescent dye and tested in-house for direct flow cytometric analysis in mouse cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated VCAM-1 (D8U5V) Rabbit mAb (Mouse Specific) #39036.

VCAM-1 (vascular cell adhesion molecule-1) is a transmembrane glycoprotein containing multiple amino-terminal extracellular Ig-like domains, a transmembrane domain, and a short carboxy-terminal cytoplasmic domain (1). Alternative splicing generates two isoforms of VCAM-1 (2). The role of VCAM-1 during infection and inflammatory diseases is well characterized. Expression of VCAM-1 is induced in endothelial cells by inflammatory cytokines including TNF-α and IL-1β (1). VCAM-1 on endothelial cells interacts with the integrin VLA-4 (α4β1) on leukocytes to mediate migration of circulating leukocytes from the blood across the endothelium and into tissues (3).

  1. Osborn, L. et al. (1989) Cell 59, 1203-11.
  2. Hession, C. et al. (1991) J Biol Chem 266, 6682-5.
  3. Elices, M.J. et al. (1990) Cell 60, 577-84.
Entrez-Gene Id
22329
Swiss-Prot Acc.
P29533
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.
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 outlicensing@lifetech.com.

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Product # Size Price
15631S
100 µl  (50 tests) N/A

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