Flow cytometric analysis of ACHN cells (blue) and Ramos cells (green) using Syk (4D10) Mouse mAb (Alexa Fluor® 488 Conjugate) #20767 (solid lines) or a concentration-matched Mouse (E5Y6Q) mAb IgG2a Isotype Control (Alexa Fluor® 488 Conjugate) (dashed lines).
This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 488 fluorescent dye under optimal conditions and tested in-house for direct flow cytometric analysis of human cells.
Note: This control antibody must be diluted to the same concentration (not dilution) as the specific antibody in analysis. See Directions for Use.
Directions for Use: Important! This control antibody must be diluted to the same concentration (not dilution) as the specific antibody used for analysis. Higher background fluorescence may result if excessive amounts of mouse IgG isotype control are used. Do not use this antibody at 1:50 dilution in the same way as recommended for other CST Alexa Fluor® conjugated antibodies.
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
Mouse (E5Y6Q) mAb IgG2a Isotype Control (Alexa Fluor® 488 Conjugate) is not directed against any known antigen. It functions as an isotype control for mouse IgG2a monoclonal antibodies.
Isotype control antibodies are used to estimate the nonspecific binding of target primary antibodies due to Fc receptor binding or other protein-protein interactions. An isotype control antibody should have the same immunoglobulin type and be used at the same concentration as the test antibody.