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ASC (D2W8U) Rabbit mAb (Mouse Specific) (PE Conjugate) #70891
Flow cytometric analysis of Raw 264.7 cells (blue) and J774A.1 cells (green) using ASC (D2W8U) Rabbit mAb (Mouse Specific) (PE Conjugate) (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (dashed lines).Learn more about how we get our images
Gallery: ASC (D2W8U) Rabbit mAb (Mouse Specific) (PE Conjugate) #70891
Flow Cytometry, Methanol Permeabilization Protocol for Direct Conjugates
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
- 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
- 16% Formaldehyde (methanol free).
- 100% methanol.
- Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.
- Collect cells by centrifugation and aspirate supernatant.
- Resuspend cells in 0.5-1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
- Fix for 15 min at room temperature.
- Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS.
- Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
- Incubate 30 min on ice.
- Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.
- Aliquot desired number of cells into tubes or wells.
- Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
- Resuspend cells in 100 µl of diluted antibody conjugate (prepared in incubation buffer at the recommended dilution).
- Incubate for 1 hr at room temperature. Protect from light.
- Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
- 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
- Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
- Incubate for at least 5 min at room temperature.
- Analyze cells in DNA staining solution on flow cytometer.
posted July 2009
revised June 2017
ASC (D2W8U) Rabbit mAb (Mouse Specific) (PE Conjugate) recognizes endogenous levels of total ASC protein.Species Reactivity: Mouse
Monoclonal antibody is produced by immunizing animals with recombinant mouse ASC protein.
This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometric analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated ASC (D2W8U) Rabbit mAb (Mouse Specific) #67824.
TMS1 (target of methylation-induced silencing)/ASC (apoptosis-associated speck-like protein containing a CARD), also referred to as PYCARD and CARD5, is a 22-kDa pro-apoptotic protein containing an N-terminal pyrin domain (PYD) and a C-terminal caspase recruitment domain (CARD) (1-2). The TMS1 gene was originally found to be aberrantly methylated and silenced in breast cancer cells (2), and has since been found to be silenced in a number of other cancers, including ovarian cancer (3), glioblastoma (4), melanoma (5), gastric cancer (6), lung cancer (7), and prostate cancer (8). Expression of TMS1 can be induced by pro-apoptotic/inflammatory stimuli (9). During apoptosis TMS1 is re-distributed from the cytosol to the mitochondria and associates with mitochondrial Bax to trigger cytochrome c release and subsequent apoptosis (10). TMS1 has also been found to be a critical component of inflammatory signaling where it associates with and activates caspase-1 in response to pro-inflammatory signals (11).
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.