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- Additional protein information
- Analytical tools
CellSimple™ Cellular Stress Antibody Assay Kit #29213
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CellSimple™ cell-based analysis of untreated (left) or UV treated (right) HeLa cells using CellSimple™ Cellular Stress Antibody Assay Kit. Data was collected in both green (525/45 nm) and red (561 nm LP) channels and analyzed on the Open Flow application. Dot plots represent mean fluorescence intensity (MFI) of Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (Alexa Fluor® 488 Conjugate) #9719 (x-axis) and Phospho p38 MAPK (Thr180/Tyr182) (3D7) Rabbit mAb (PE Conjugate) #6908 (y-axis). Instrument screen shots are shown.Learn more about how we get our images
Gallery: CellSimple™ Cellular Stress Antibody Assay Kit #29213
- CellSimple™ cell-based analysis of untreated (left) or UV treated (right) HeLa cells using CellSimple™ Cellular Stress Antibody Assay Kit. Data was collected in both green (525/45 nm) and red (561 nm LP) channels and analyzed on the Open Flow application. Dot plots represent mean fluorescence intensity (MFI) of Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (Alexa Fluor® 488 Conjugate) #9719 (x-axis) and Phospho p38 MAPK (Thr180/Tyr182) (3D7) Rabbit mAb (PE Conjugate) #6908 (y-axis). Instrument screen shots are shown.
Immunostaining Protocol for CellSimple™ Antibody-based Assay for Kits
These kits were specially designed for use with the CellSimple™ Cell Analyzer. However, they may also be used with a flow cytometer or plate reader capable of providing excitation between 480 nm and 490 nm and detecting fluorescent emission between 520 nm and 590 nm.
B. Kit components:
- Antibody pair (kit specific)
- 16% Formaldehyde (Methanol-free)
NOTE: The screw cap allows for the entire vial contents to be used at once. To extend the product's shelf life, small volumes should be extracted by piercing the silicone top with a needle and syringe. Store protected from light and use within one month after opening.
- Phosphate Buffered Saline (PBS-20X)
C. Additional reagents needed, but not supplied.
- 90% methanol
- Bovine Serum Albumin #9998 or equivalent
- Reverse osmosis/deionized (RO/DI) water or equivalent
D. Reagent preparation
- 1X PBS: To prepare 500 ml 1X PBS add 25 ml PBS-20X to 475 ml RO/DI water, mix.
- Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin in 100 ml 1X PBS. Store at 4°C.
- Collect cells by centrifugation and aspirate supernatant. For adherent cells, remove cells from the plate or flask by trypsinization followed by trypsin neutralization with medium that contains 10% fetal bovine serum.
- Resuspend cells in 0.5 - 1 ml 1X PBS to 0.5 X 106 - 5 X 106 cells/ml by gently pipetting up and down. Add 16% Formaldehyde to obtain a final concentration of 4% and vortex gently (for example 335 µl of 16% Formaldehyde added to 1 ml of cell suspension).
- Fix for 10 min at 37°C.
- Chill tubes on ice for 1 minute.
- Spin down the cells at 400 x g for 5 minutes in a chilled microcentrifuge.
- Carefully remove the supernatant containing formaldehyde into a hazardous waste receptacle.
- Permeabilize cells by slowly adding 1 - 1.5 ml of ice-cold 90% methanol. Pipette up and down several times to ensure uniform cell suspension.
- Incubate 30 min on ice.
- Proceed with immunostaining or store cells at -20°C in 90% methanol.
- Aliquot 0.5 - 1 x 106 cells into each assay tube.
- Wash the cells twice in 1 ml incubation buffer by centrifugation at 400 x g for 5 minutes.
- Dilute each of the two fluorochrome-conjugated antibodies 1:50 in incubation buffer. Prepare enough diluted antibody mix to resuspend each of your test samples in 25 µl. For example, for 3 test samples add 1.5 µl of antibody mix to 75 µl of incubation buffer. Note: the antibodies are mixed together to allow a simultaneous detection in both the green and the red channels.
- Add 25 µl of the diluted antibody mix to each test sample and gently pipette up and down a few times to ensure even cell suspension.
- Incubate for 1 hour at room temperature with occasional gentle mixing.
- Wash once by centrifugation using incubation buffer.
- Resuspend cells in 0.1 - 0.2 ml PBS and analyze 75 µl on CellSimple™ Cell Analyzer using the Open Flow Cytometry application and using both Green (525/45 nm) and Red (561 nm LP) channels. For more information on how to use the Open Flow Cytometry application and detailed instructions on how to operate the CellSimple™ Cell Analyzer please refer to the CellSimple™ user guide.
posted June 2016
revised November 2016
|Product Includes||Quantity (with Count)|
|Phospho-p38 MAPK (Thr180/Tyr182) (3D7) Rabbit mAb (PE Conjugate) 6908||1 x 25 µl|
|Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (Alexa Fluor® 488 Conjugate) 9719||1 x 25 µl|
|16% Formaldehyde, Methanol-Free 12606||2 x 10 ml|
|Phosphate Buffered Saline (PBS-20X) 9808||1 x 25 ml|
The CellSimple™ Cellular Stress Antibody Assay Kit is designed for use with the CellSimple™ Cell Analyzer. The kits allows for the quick and easy measurement of the extent of cellular stress or DNA damage under given experimental conditions.
Phospho-p38 MAPK (Thr180/Tyr182) (3D7) Rabbit mAb (PE conjugate) recognizes endogenous levels of p38 MAPK only when dually phosphorylated at
Thr180/Tyr182. This antibody does not cross-react with other MAPK family members. Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (Alexa Fluor® 488 Conjugate) recognizes H2A.X only when phosphorylated at Ser139. This antibody does not cross react with other phosphorylated histone proteins.Species Reactivity: Human, Mouse
The CellSimple™ Cell Analyzer is a benchtop instrument that utilizes a disposable thin-film cassette and a combination of a 488 nm laser, two photomultiplier tubes (525/45 nm and 561 nm LP filters), Coulter Principle-based cell measurements, and on-board software to provide easy-to-run applications and data analysis. Data acquisition occurs within approximately 10 seconds per test. The instrument relies on disposable cassettes for sample handling, which alleviates the need for flow cell cleaning and fluidics maintenance and the instrument is small enough to be portable between the lab bench and the hood. Applications include quantitative assessments of cell viability, apoptosis, other labeled antibody markers and single and multiplexed bead-based assays for protein and cellular analysis.
p38 MAP kinase (MAPK) is activated by a variety of cellular stresses including osmotic shock, inflammatory cytokines, lipopolysaccharide (LPS), UV light, and growth factors. p38 MAPK is activated by phosphorylation at Thr180 and Tyr182 by various MAPK kinases. Once activated the p38 MAPK phosphorylates a wide spectrum of cellular targets to help the cells adapt and cope with noxious conditions (1). Histone H2A.X is required for checkpoint-mediated cell cycle arrest and DNA repair following double-stranded DNA breaks. DNA damage, caused by ionizing radiation, UV-light, or radiomimetic agents results in rapid phosphorylation of H2A.X at Ser139. This very early event in the DNA-damage response is required for recruitment of a multitude of DNA-damage response proteins. In addition to its role in DNA-damage repair, H2A.X is required for DNA fragmentation during apoptosis and is phosphorylated by various kinases in response to apoptotic signals (2).
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. CellSimple is a trademark of Cell Signaling Technology, Inc. XP is a registered trademark of Cell Signaling Technology, Inc. Alexa Fluor is a registered trademark of Life Technologies Corporation. The Alexa Fluor dye conjugates in this product are sold under license from Life Technologies Corporation, for research use only excluding use in combination with DNA microarrays and high content screening (HCS). This product is provided under an intellectual property license from Life Technologies Corporation. The transfer of this product is contingent on the buyer using the purchased product solely in research, including use with HCS or other automated imaging applications but excluding use in combination with DNA microarrays. The buyer must not sell or otherwise transfer this product or its components for (a) diagnostic, therapeutic or prophylactic purposes; (b) testing, analysis or screening services, or information in return for compensation on a per-test basis; (c) manufacturing or quality assurance or quality control, or (d) resale, whether or not resold for use in research. For information on purchasing a license to this product for purposes other than as described above, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or email@example.com.