|Flow Cytometry (Fixed/Permeabilized)||1:50|
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
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human ALDH1A1 protein.
The aldehyde dehydrogenase family is a large group of enzymes that oxidize aldehydes formed through metabolic processes to their carboxylic acids (1). ALDH1A1 is a liver cytosolic isoform of acetaldehyde dehydrogenase and is involved in the major pathway of alcohol metabolism along with alcohol dehydrogenase (2). ALDH1A1 is also known as retinal dehydrogenase 1 and is involved in retinol metabolism, converting retinol to retinoic acid (3). Recent studies suggest that control of retinoid signaling through ALDH1A1 may influence hematopoietic stem cell differentiation (4). There has been recent interest in ALDH1 isoforms as predictive biomarkers in disease. Several studies have suggested that ALDH1A1 is a potential marker for cancer stem cells and chemoresistance in several tumor types, such as melanoma (5), lung cancer (6), and glioblastoma (7).
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