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Dec 31st Santa Cruz will discontinue a
large number of polyclonal products as
a result of the USDA settlement that was
made public May 19th.

Find CST Equivalent

To Purchase # 75173S

75173S 1 Kit (50 assays) $409.00.0
$0.00

Questions?

Find answers on our FAQs page.

ANSWERS  

PhosphoSitePlus® Resource

  • Additional protein information
  • Analytical tools

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REACTIVITY SENSITIVITY MW (kDa) SOURCE
H
Page

Immunostaining Protocol for CellSimple™ Antibody-based Assay for Kits

A. Instrumentation:

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

  1. 1X PBS: To prepare 500 ml 1X PBS add 25 ml PBS-20X to 475 ml RO/DI water, mix.
  2. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin in 100 ml 1X PBS. Store at 4°C.

E. Fixation

  1. 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.
  2. 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).
  3. Fix for 10 min at 37°C.
  4. Chill tubes on ice for 1 minute.
  5. Spin down the cells at 400 x g for 5 minutes in a chilled microcentrifuge.
  6. Carefully remove the supernatant containing formaldehyde into a hazardous waste receptacle.

F. Permeabilization

  1. 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.
  2. Incubate 30 min on ice.
  3. Proceed with immunostaining or store cells at -20°C in 90% methanol.

G. Immunostaining

  1. Aliquot 0.5 - 1 x 106 cells into each assay tube.
  2. Wash the cells twice in 1 ml incubation buffer by centrifugation at 400 x g for 5 minutes.
  3. 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.
  4. 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.
  5. Incubate for 1 hour at room temperature with occasional gentle mixing.
  6. Wash once by centrifugation using incubation buffer.
  7. 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 Description

CellSimple™ Cleaved Caspase-3 and Phospho-Histone H3 Antibody Assay Kit is a fluorescent assay designed for use with the CellSimple™ Cell Analyzer. The kit includes Cleaved Caspase-3 (Asp175) (D3E9) Rabbit mAb (PE Conjugate) #12768 and Phospho-Histone H3 (Ser10) (D2C8) XP® Rabbit mAb (Alexa Fluor® 488) #3465. The kit allows for the quick and easy assessment of the levels of mitotic cells undergoing apoptosis.


Product Usage Information

Storage: The conjugated antibodies in this kit are 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 antibodies. Protect from light. Do not freeze. The remaining components of the kit should be stored at the recommended temperature upon receipt. All components in this kit are stable for at least 12 months when stored at the recommended temperature and left unused.

Specificity / Sensitivity

Antibodies provided in the CellSimple™ Cleaved Caspase-3 and Phospho-Histone H3 Antibody Assay Kit detect endogenous levels of their respective target.


Species Reactivity: Human

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.


Caspase-3 (CPP-32, Apoptain, Yama, SCA-1) is a critical executioner of apoptosis, as it is either partially or totally responsible for the proteolytic cleavage of many key proteins, such as the nuclear enzyme poly (ADP-ribose) polymerase (PARP) (1). Activation of caspase-3 requires proteolytic processing of its inactive zymogen into activated p17 and p12 fragments. Cleavage of caspase-3 requires the aspartic acid residue at the P1 position (2). Modulation of chromatin structure plays an important role in the regulation of transcription in eukaryotes. The nucleosome, made up of DNA wound around eight core histone proteins (two each of H2A, H2B, H3, and H4), is the primary building block of chromatin (3). The amino-terminal tails of core histones undergo various post-translational modifications, including acetylation, phosphorylation, methylation, and ubiquitination (4-6). These modifications occur in response to various stimuli and have a direct effect on the accessibility of chromatin to transcription factors and, therefore, gene expression (7). Phosphorylation at Ser10 of histone H3 is tightly correlated with chromosome condensation during both mitosis and meiosis (8).


1.  Chen, D.L. et al. (2015) Mol Imaging Biol 17, 384-93.

2.  Konstantinidou, A.E. et al. (2007) Apoptosis 12, 695-705.

3.  He, L. et al. (2005) J Immunol Methods 304, 43-59.

4.  Rayzman, V.M. and Sentry, J.W. (2006) Hum Antibodies 15, 71-80.

5.  Hirata, A. et al. (2004) J Histochem Cytochem 52, 1503-9.

6.  Juan, G. et al. (1998) Cytometry 32, 71-7.

7.  Bernstein, B.E. and Schreiber, S.L. (2002) Chem Biol 9, 1167-73.

8.  Goto, H. et al. (1999) J Biol Chem 274, 25543-9.



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.
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 outlicensing@lifetech.com.

75173
CellSimple™ Cleaved Caspase-3 and Phospho-Histone H3 Antibody Assay Kit