04511 Cellstain double staining kit
Application
Double Staining Kit is utilized for simultaneous fluorescence staining of viable and dead cells. This kit
contains Calcein-AM and Propidium Iodide (PI) solutions, which stain viable and dead cells,
respectively. Calcein-AM, acetoxymethyl ester of calcein, is highly lipophilic and cell membrane
permeable. Though Calcein-AM itself is not a fluorescent molecule, the calcein generated from Calcein-
AM by esterase in a viable cell emits strong green fluorescence (excitation: 490 nm, emission: 515
nm). Therefore, Calcein-AM only stains viable cells. On the other hand, PI, a nuclei staining dye,
cannot pass through a viable cell membrane. It reaches the nucleus by passing through disordered
areas of dead cell membrane, and intercalates with the DNA double helix of the cell to emit red
fluorescence (excitation: 535 nm, emmision: 617 nm). Since both calcein and PI-DNA can be excited
with 490 nm, simultaneous monitoring of viable and dead cells is possible with a fluorescence
microscope. With 545 nm excitation, only dead cells can be observed. Since optimal staining conditions
differ from cell line to cell line, we recommend that a suitable concentration of PI and Calcein-AM be
individually determined. Please note that PI is suspected to be highly carcinogenic; careful handling is
required.
Content
Solution A: 4 vials
Solution B: 1 vial
Methods
1. Add 10 µl Solution A and 5 µl Solution B to 5 ml PBS to prepare assay solution.a)
2. Wash cells with PBS several times to remove residual esterase activity.
3. Prepare a cell suspension with PBS in which the cell density is 1x105 to 1x106 cells/ml.
4. Add 100 µl of assay solution to cells and incubate the mixture at 37 ºC for 15 min.
5. Detect fluorescence using a fluorescence microscope with 490 nm excitation for simultaneous
monitoring of viable and dead cells. With 545 nm excitation, only dead cells can be observed.
a) The concentration of each reagent should be optimized. Following steps may be necessary
to determine the suitable concentration of each reagent:
1. Prepare dead cells by 10 min incubation in 0.1% saponin or 0.1-0.5% digitonin or by 30
min incubation in 70% ethanol.
2. Stain dead cells with 0.1-10 µM PI solution to find a PI concentration that stain nucleus
only, does not stain cytosol.
3. Stain dead cells with 0.1-10 µM Calcein-AM solution to find a Calcein-AM concentration
that does not stain cytosol. Then stain viable cells with that Calcein-AM solution to check
whether the viable cell can be stained.
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Storage
Reagent solution is stable for more than 12 months at -20 °C with protection from light and moisture.
Since the buffer solution of Calcein-AM is gradually hydrolyzed to generate fluorescent Calcein, the
working
solution is not storable. Close the bottle cap tightly after using a portion of Calcein AM solution to avoid
moisture.
HeLa cell, incubated with assay solution for 15 min.
A) viable cell
B) dead cell
References
1. Kimura, K., et al., Neurosci. Lett., 208, 53 (1998).
2. Matsuse, S., et al., J. Clin. Pathol., in press (1998).
3. Shimokawa, I., et al., J. Geronto., 51a, b49 (1998).
4. Yoshida, S., et al., Clin. Nephrol., 49, 273 (1998).
5. Tominaga, H., et al., Anal. Commun., 36, 47 (1999).
Precautions and Disclaimer
This product is for R&D use only, not for drug, household, or other uses. Please consult the Material
Safety Data Sheet for information regarding hazards and safe handling practices.
The vibrant M and Sigma-Aldrich are trademarks of Merck KGaA, Darmstadt, Germany or its affiliates.
Detailed information on trademarks is available via publicly accessible resources.
© 2018 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.
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