Assessment of cell viability is a critical step during the evaluation of novel drug treatments and therapies for potential cytotoxic properties. ICT’s Basic Calcein AM Cell Viability kit allows for easy assessment of cell viability in a sample.
To use Calcein AM, simply add the reagent directly to the cell sample, incubate, and analyze (no wash steps necessary). 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.
Samples can be analyzed using a flow cytometer, fluorescence plate reader, 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 to prepare 2 mM stock solution.
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. Analzye with a flow cytometer, fluorescence microscope, or fluorescence plate reader. Calcein AM excites at 494 nm and emits at 520 nm.
Microscopy Analysis of Live Jurkat Cells Stained with Calcein AM
Untreated Jurkat suspension cells were stained with 1 µM Calcein AM for 60 minutes at 37°C to detect live cells. The majority of the cells imaged were considered to be live and healthy.
Panel A reveals green fluorescence-stained live cells. Panel B shows a corresponding differential interference contrast (DIC) image, which reveals cell morphology.
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 fluorescence plate reader data.
• Reagent name: Calcein AM
• Excitation/Emission: 494 nm / 520 nm
• Method of Analysis: Flow cytometer, fluorescence microscope, fluorescence plate reader
• 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
• Hoechst 33342, 1 vial, #639
• 10X Cellular Assay Buffer, 60 mL, 1 bottle, #6695
• Kit Manual