Confocal immunofluorescent analysis of mouse astrocytes, containing either a knock-in FLAG-tagged K27M mutant histone H3.3 gene (left, positive) or a knock-in FLAG-tagged wild-type histone H3.3 gene (center, negative), and HeLa cells (right, negative), using Histone H3 (K27M Mutant Specific) (D3B5T) Rabbit mAb (Alexa Fluor® 488 Conjugate) (green) and β-Actin (8H10D10) Mouse mAb #3700 (red).
Flow cytometric analysis of mouse astrocytes containing either a knock-in FLAG-tagged K27M mutant histone H3.3 gene (green) or a knock-in FLAG-tagged wild-type histone H3.3 gene (blue), using Histone H3 (K27M Mutant Specific) (D3B5T) Rabbit mAb (Alexa Fluor® 488 Conjugate) (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (Alexa Fluor® 488 Conjugate) #2975 (dashed lines).
This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 488 fluorescent dye and tested in-house for direct flow cytometric and immunofluorescent analysis in mouse cells and immunofluorescent analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated Histone H3 (K27M Mutant Specific) (D3B5T) Rabbit mAb #74829.
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.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
posted December 2010
Protocol Id: 221
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
Histone H3 (K27M Mutant Specific) (D3B5T) Rabbit mAb (Alexa Fluor® 488 Conjugate) recognizes endogenous levels of K27M mutant histone H3.1, H3.2, and H3.3 proteins. The antibody may cross-react with wild-type histone H3.1, 3.2, and 3.3 when used at a high concentration. Careful titration of this antibody may be required to obtain optimal specificity.
Rat, Xenopus, Pig
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to K27M mutant sequence of human histone H3.3 protein.
Diffuse intrinsic pontine glioma (DIPG) is an aggressive brainstem astrocyte tumor arising mostly in children, leading to a long-term survival rate of less than 10%. Multiple whole-genome sequencing studies of DIPG patients identified commonly occurring mutations in the H3F3A gene encoding histone H3.3. One of these mutations, a lysine to methionine amino acid substitution (K27M), is found in up to 78% of DIPGs and 22% of non-brainstem pediatric gliomas (1-3). This mutation is associated with poor prognosis, with a mean survival time of 0.73 years for patients with the K27M mutation versus 4.6 years for patients without the mutation (1-3). Expression of the K27M mutant histone H3 is accompanied by a dramatic reduction in the levels of polycomb repressive complex 2 (PRC2)-mediated trimethylation of histone H3, changes in the distribution of PRC2 on the genome, and altered expression of genes associated with various cancer pathways (4-6).
Explore pathways + proteins related to this product.
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