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To Purchase # 62734S

62734S 1 Kit (50 assays) $369.00
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PhosphoSitePlus® Resource

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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)
  • 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 1 L 1X PBS add 50 ml PBS-20X to 950 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

Product Includes Quantity (with Count) Solution Color
Oct-4A (C30A3) Rabbit mAb (Alexa Fluor® 488 Conjugate) 5177 1 x 25 µl
Nanog (D73G4) XP® Rabbit mAb (PE Conjugate) 14955 1 x 25 µl
16% Formaldehyde, Methanol-Free 2 x 10 ml
Phosphate Buffered Saline (PBS-20X) 9808 1 x 25 ml

Product Description

CellSimple™ Stem Cell Pluripotency Antibody Assay Kit is designed for use with the CellSimple™ Cell Analyzer. The kit allows for the quick and easy measurement of Nanog and Oct-4A, two key proteins that are specifically expressed in human pluripotent cells. The kit includes Nanog (D73G4) XP® Rabbit mAb (PE Conjugate) #14955 and Oct-4A (C30A3) Rabbit mAb (Alexa Fluor® 488 Conjugate) #5177.


Specificity / Sensitivity

Antibodies provided in the CellSimple™ Stem Cell Pluripotency 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.


Pluripotency is the ability of a cell to differentiate into cell types of the three germ layers, the endoderm, ectoderm, and mesoderm. It is a property shared by embryonic stem cells, embryonic carcinoma and induced pluripotent cells. Oct-4 and Nanog together with Sox2 are key transcriptional regulators that are highly expressed in pluripotent cells (1). Together they form a transcriptional network that maintains cells in a pluripotent state (2,3). Overexpression of Oct-4 and Sox2 along with Klf4 and c- Myc can induce pluripotency in both mouse and human somatic cells, highlighting their roles as key regulators of the transcriptional network necessary for renewal and pluripotency (4-5). Upon differentiation of pluripotent cultures, expression of Oct-4 and Nanog is induced.


1.  Pesce, M. and Schöler, H.R. (2001) Stem Cells 19, 271-8.

2.  Pan, G. and Thomson, J.A. (2007) Cell Res 17, 42-9.

3.  Takahashi, K. and Yamanaka, S. (2006) Cell 126, 663-76.

4.  Yu, J. et al. (2007) Science 318, 1917-20.

5.  Okita, K. et al. (2007) Nature 448, 313-7.



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.
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).

62734
CellSimple™ Stem Cell Pluripotency Antibody Assay Kit