Experimental Protocol for Calcein AM Assay

by K Chico

Simultaneous imaging of live and apoptotic HeLa cells labeled using calcein AM (green) and Annexin V-iFluor® 647 conjugate (red).

The calcein AM viability assay is a simple sensitive assay for measuring cell viability, cell proliferation , or to assess cytotoxicity in adherent cells or those in suspension. Calcein AM interacts with intracellular esterases to produce a hydrophilic, strongly fluorescent compound that is retained in the cytoplasm. Analysis can be performed at an Ex/Em = 490/525 nm in a fluorescent microplate reader or under an appropriate channel in a flow cytometer. Other calceins may require other spectral settings outside of the typical green wavelengths. The measured fluorescence intensity is proportional to the number of viable cells within a sample.

Materials and Equipment

Calcein AM
Calcein AM assay buffer
Centrifuge
Culture microplates
PBS
Lab grade deionized water
Cell culture medium
CO2 incubator
DMSO
FACS tubes
Ethanol/formaldehyde
Fluorescence microplate reader
Flow cytometer
96-well flat-bottom, black plate

Reagent Preparation

Calcein AM assay buffer: if not supplied ready to use, dilute in distilled or deionized water as instructed. A common option is Hanks and Hepes buffer, but others may be employed based on experimental requirements. A table of recipes and preparation instructions for various buffers is available here.

Calcein AM stock solution: resuspend calcein AM in an appropriate amount of anhydrous DMSO to make a stock solution. For most cell lines, Calcein AM at the final concentration of 4 to 5 µM is recommended. The nonionic detergent Pluronic® F-127 at a final concentration of 0.02% is sometimes used to increase the aqueous solubility of AM esters.

Table 1. Common stock solution preparation.

▲ ▼	50 µg ▲ ▼	100 µg ▲ ▼	500 µg ▲ ▼	1 mg ▲ ▼	5 mg ▲ ▼
1 mM	50.258 µL	100.517 µL	502.583 µL	1.005 mL	5.026 mL
5 mM	10.052 µL	20.103 µL	100.517 µL	201.033 µL	1.005 mL
10 mM	5.026 µL	10.052 µL	50.258 µL	100.517 µL	1.005 mL

Note:

Aliquots can be stored at -20 ℃ and should be used within two months. Immediately prior to use, dilute the calcein AM stock solution in the calcein AM assay buffer, preparing at least enough for all wells.
The optimal concentration of calcein AM may vary depending on cell type. In general, this should be empirically determined.

Table 2. Specifications for calcein AM and calcein AM derivatives.

Product ▲ ▼	Ex (nm) ▲ ▼	Em (nm) ▲ ▼	Spectrum ▲ ▼	Unit Size ▲ ▼	Cat No. ▲ ▼
Calcein Blue, AM CAS 168482-84-6	354 nm	441 nm		1 mg	22007
Calcein UltraBlue™	359 nm	458 nm		1 mg	21908
CytoCalcein™ Violet 450 Excited at 405 nm	406 nm	445 nm		1 mg	22012
CytoCalcein™ Violet 500 Excited at 405 nm	420 nm	505 nm		1 mg	22013
Calcein, AM CAS 148504-34-1	501 nm	521 nm		1 mg	22002
Calcein, AM UltraPure grade CAS 148504-34-1	501 nm	521 nm		1 mg	22003
Calcein, AM UltraPure grade CAS 148504-34-1	501 nm	521 nm		20x50 µg	22004
Calcein Orange™ diacetate	531 nm	545 nm		1 mg	22009
Calcein Red™ AM	562 nm	576 nm		1 mg	21900
Calcein Deep Red™ AM ester	643 nm	663 nm		1 mg	22011

Fluorescence Microplate

Detection of Jurkat cell viability using calcein Blue AM. Jurkat cells were treated with staurosporine and stained with Calcein Blue AM (blue), annexin V iFluor® 488 (green), and 7-AAD (red).

Cell Preparation

Grow cells as instructed in an appropriate 96-well plate.
Note: Clear plates can be used to ensure cell adhesion, but will increase background fluorescence and may reduce assay sensitivity. If using a clear plate, samples can be transferred to a solid black plate prior to reading. In general, AAT Bioquest recommends using a black wall/clear bottom plate, and setting the instrument to bottom read mode.
For adherent cells, carefully aspirate the media.
For suspension cells, spin the 96-well plate at 1,000 g for 5 minutes at 4°C and carefully aspirate the media.

Protocol

For the most reproducible assay, fresh samples are recommended. Snap frozen samples are the second best alternative, though it is important to avoid multiple freeze-thaws. Addition of a protease inhibitor may be necessary for certain samples.

Plate cells in a 96-well plate with the densities from 1e3 to 5e5 cells/mL.
- Prepare duplicates and/or triplicates as desired.
- Cell concentrations must be optimized to ensure the best dynamic range.
Optional: It may be necessary to remove leftover media in the supernatant, as phenol red and serum can interfere with the sensitivity of the assay. If so:
1. Centrifuge the plate at 250 G for 5 minutes and remove the supernatant.
2. Wash cells with an appropriate amount of fresh assay buffer or PBS.
3. Repeat centrifugation and aspiration steps.
Add an appropriate amount of fresh assay buffer to each well.
Add an appropriate amount of fresh calcein AM stock solution to each well.
Incubate for 30 minutes at 37°C, or as otherwise instructed or optimized. 20 minutes to 1 hour is a typical range of time. Decreasing the loading temperature may reduce compartmentalization.
Note: Incubation time should be empirically determined based on cell type and concentration.
Wash cells in indicator-free buffer (containing an anion transporter inhibitor, if applicable) to remove excess probe.
Read fluorescence in the microplate reader at Ex/Em 490/525 nm cutoff 515nm, or other spectral points depending on if other calcein variants in alternative colors are used.

Flow Cytometry

Flow Cytometry Analysis of Jurkat cells stained with Calcein Deep Red™ AM ester. Jurkat cells were washed once with HH buffer and stained with 2 uM Calcein Deep Red™ AM ester in HH with 0.02% PF-127 and 1mM PBC for 30 minutes at 37C incubator. Cells were then washed with HH buffer and resuspended in HH buffer. The fluorescence intensities of Live cells (healthy, Red) and Dead cells (treated in 55°C water bath for 30 minutes, Green) were measured with NovoCyte 3000 flow cytometer using blue laser APC emission channel.

Cell Preparation

Ideally, the cell concentration should be between 3e5 to 5e5 cells/mL.

Note: The concentration should remain <106 cells/mL, as cells cultivated in excess may naturally enter apoptosis and/or may not be analyzed efficiently by the flow cytometer.

Positive control: live, non-treated cells grown in a normal culture environment.
Negative control: Cells that undergo induced death. This population can be achieved by exposing a sample to:

90% Ethanol for 30-60 seconds at 37°C; or
3% Formaldehyde for 30 minutes on ice, followed by a wash step with PBS, and resuspension in the assay buffer.

Protocol

Transfer an appropriate amount of cells into fresh FACS tubes, including repeats as desired. The following controls and samples should be prepared in separate tubes:
- Unlabeled live cells
- Unlabeled dead cells
- Calcein AM stained live cells
- Calcein AM stained dead cells
Add an appropriate amount of the reconstituted calcein AM stock solution to each respective tube.
Mix the cells gently through tapping the tube or pipetting.
Incubate cells, protected from light, at 37 °C.
Note: Incubation period may range from 30 minutes, depending, and can be optimized for each cell line and experimental condition. With longer incubation times, cells may settle on the bottom of the tubes. Gentle resuspension can be performed by gently swirling tubes or pipetting the solution periodically to ensure an even distribution of calcein AM throughout the staining step.
Measure fluorescence of each tube on the flow cytometer equipped with appropriate lasers. For calcein AM, this will require a blue laser, and the instrument should be set to the FITC channel (530/30 filter set).
- Live cells will fluoresce due to the presence of active esterases capable of cleaving calcein AM.
- Dead cells will not fluoresce due to the absence of active esterases, leaving calcein AM in its uncleaved form.
Note: Wash steps are not required. Any calcein AM that has not been cleaved by intracellular esterases will be nonfluorescent.

Selection Guide for Calcein Viability Dyes

Dye		Calcein Blue	CytoCalcein™ Violet 450	Calcein UltraBlue™	CytoCalcein™ Violet 500	Calcein UltraGreen™	Calcein	Calcein Orange™	Calcein Red™	Calcein Deep Red™
Principle		In live cells, non-fluorescent calcein AM is converted to a fluorescent calcein product after AM ester hydrolysis by intracellular esterases.
Readout		Live cells are highly fluorescent and easily distinguishable from dead cells, which are non-fluorescent.
Laser(nm)		UV (350)	Violet (405)	UV (350)	Violet (405)	Blue (488)	Blue (488)	Green (532)	Yellow (561-568)	Red (633-647)
Ex/Em(nm)		354/441	406/445	359/458	420/505	492/514	501/521	531/545	562/576	643/663
Multiplexable		Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes
Dye Compatibility		Compatible with green and red fluorescent proteins, indicators, and other probes	Compatible with green and red fluorescent proteins, indicators, and other probes	Compatible with green and red fluorescent proteins, indicators, and other probes	Compatible with blue and red fluorescent proteins, indicators, and other probes	Compatible with blue and red fluorescent proteins, indicators, and other probes	Compatible with blue and red fluorescent proteins, indicators, and other probes	Compatible with blue and red fluorescent proteins, indicators, and other probes	Compatible with blue and green fluorescent proteins, indicators, and other probes	Compatible with blue and green fluorescent proteins, indicators, and other probes
Sample Type		Live Cells
Platform		Fluorescence Microscope, Fluorescence Microplate Reader, Flow Cytometer
Cat No.	AM Ester	22007	22012	21908	22013	21905	22003	22009	21900	22011
Cat No.	Reference Dye	22006		21909			22001	22008	21901	21902
Spectrum

References

A Protocol for a High-Throughput Multiplex Cell Viability Assay
An Improved Flow Cytometry-Based Natural Killer Cytotoxicity Assay Involving Calcein AM Staining of Effector Cells
Staining of fluorogold-prelabeled retinal ganglion cells with calcein-AM: A new method for assessing cell vitality

Original created on October 24, 2023, last updated on August 20, 2024
Tagged under: calcein, cell viability, protocol