Assessment of cell viability is a critical step during the evaluation of novel drug treatments and therapies for potential cytotoxic properties. ICT’s Advanced Calcein AM Cell Viability Kit combines Calcein AM with 7-AAD to allow for easy and simultaneous labeling of live, membrane compromised, and dead cells within a single sample.
Calcein AM is a membrane permeant, fluorogenic, reagent widely recognized for its utility in assessing the relative cell viability status of different cell populations. Calcein AM’s overall hydrophobic nature allows it to readily traverse the lipid bilayer structure of the cell membrane in a concentration gradient-dependent manner. Once inside the cell, the hydrophobic and non-fluorescent Calcein AM is quickly hydrolyzed by intracellular esterases that are active in live cells. This leads to the cleavage and removal of two non-polar acetoxymethyl ester (AM) groups. Once the AM groups have been cleaved, the resulting polar (hydrophilic) and now fluorescence-capable Calcein dye molecule is efficiently retained within the confines of the cell membrane. Polar dye molecules will naturally be excluded from passive diffusion back out of the cell again due to the hydrophobic lipid bilayer composition of the cell membrane. Dead cells lack active esterases and do not cleave Calcein AM.
Loss of cell membrane integrity, often indicative of necrosis or late stage apoptosis, can be detected using the vital staining dye, 7-AAD, a red fluorescing live/dead stain. This dye easily penetrates cell membrane-compromised cells, binding tightly to GC rich regions of DNA. Combining these two different types of fluorescent cell-status-indicator reagents within a single test allows for better resolution of the live and dead cell populations by making it possible to identify the percentage of cells that are 7-AAD positive versus 7-AAD negative within the green fluorescing Calcein positive cell populations.
Samples can be analyzed using a flow cytometer or fluorescent microscope.
1. Prepare samples and controls.
2. Dilute 10X Cellular Assay Buffer 1:10 with diH2O.
3. Reconstitute Calcein AM with 50 µL DMSO.
4. Dilute 2 mM Calcein AM stock solution 1:5 by adding 200 µL diH2O or PBS, forming a 400 µM stock solution.
5. Stain with Calcein AM at a concentration between 1-10 µM. The ideal staining concentration can vary based on cell line, application, etc., and should be determined by the end user. The recommended sample size is 0.4 mL.
6. Add Calcein AM to each sample and mix gently. For example, to stain a 0.4 mL sized sample with 10 µM Calcein AM, add 10 µL of the 400 µM stock solution.
7. Incubate approximately 1 hour.
8. Reconstitute 7-AAD with 260 µL DMSO, forming a 200X stock concentrate.
9. Add 7-AAD to each sample at 1:200 (e.g., add 2 µL to 400 µL of sample).
10. Analzye with a flow cytometer or fluorescence microscope. Calcein AM excites at 494 nm and emits at 520 nm. 7-AAD excites at 546 nm and emits at 647 nm.
Comparison of Live and Dead Jurkat Cells Stained with Calcein AM and 7-AAD
Live (untreated) and dead (killed by 60 second exposure to 90% ethanol) Jurkat suspension cells were dually stained with 1 μM Calcein AM for 60 minutes at 37°C to detect live cells. Cells were then stained with 4 μM 7-AAD for 10 minutes on ice to detect membrane-compromised or dead cells.
Panel A reveals green fluorescence-stained live, untreated cells. Occasional dead cells were encountered, and stained red due to 7-AAD uptake. Panel B shows ethanol-killed cells that had been stained with both Calcein AM and 7-AAD. As there were only dead cells present in this sample type, all of the cells present stained red due to 7-AAD uptake, and no green cells were visible as Calcein AM remained in its non-fluorescent/uncleaved form. Panel C shows the staining results of a 50/50 mixture of the untreated and ethanol-killed cell populations.
Microscope images were obtained using a Nikon Eclipse 90i microscope with a Hamamatsu Flash 4.0 camera. Data courtesy of Dr. Kristi Strandberg, ICT 226:95.
Please see product manual for flow cytometry and additional microscopy data.
• Reagent name: Calcein AM, 7-AAD
• Excitation/Emission: 494 nm / 520 nm, 546 nm / 647 nm
• Method of Analysis: Flow cytometer, fluorescence microscope
• Types of Samples: Cell culture
• Storage: < -20°C for Calcein AM component, 2-8°C for remaining kit components
• Shipping: Ships overnight (domestic), International Priority Shipping
• Calcein AM Reagent, 1 vial, #6696
• 7-Aminoactinomycin D (7-AAD) vital dye, 0.26 mg, 2 vials, #6163
• Hoechst 33342, 1 vial, #639
• 10X Cellular Assay Buffer, 60 mL, 1 bottle, #6695
• Kit Manual