|Flow Cytometry (Fixed/Permeabilized)||1:50|
All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Triton™ X-100) #51995, 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 Triton™ X-100. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. Conduct a small-scale experiment if you are unsure.
NOTE: Count cells using a hemocytometer or alternative method.
posted January 2017
revised June 2020
Protocol Id: 1344
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly67 of human C/EBPβ protein.
C/EBPβ is a member of the transcription factor family of CCAAT/enhancer-binding proteins (C/EBPs) that are critical for cellular differentiation and function (1). There are various N-terminally truncated C/EBPβ isoforms: full-length C/EBPβ, C/EBPβ-LAP (liver-enriched activator protein), and C/EBPβ-LIP (liver-enriched inhibitory protein). In triple-negative breast cancer cells, aerobic glycolysis was shown to control the expression of C/EBPβ-LAP, which in turn stimulates the expression of granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF). These factors are secreted from the cancer cells to promote myeloid-derived suppressor cell (MDSC) development in the tumor microenvironment, thereby suppressing anti-tumor immunity (2). In addition, research studies showed that decreased C/EBPβ-LIP expression delays age-related phenotypes in mice, suggesting a potential role for C/EBPβ-LIP in aging (3).
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