Evaluation of cell viability using Calcein Blue AM
This protocol only provides a guideline, and should be modified according to your specific needs
1. Prepare dyeing solution
(1) Prepare stock solution: Prepare Calcein Blue AM stock solution with a concentration of 1-10mM in high-quality anhydrous DMSO. Notice: Aliquot the unused stock solution and store it in the dark at -20°C or -80°C to avoid repeated freezing and thawing.
(2) Prepare working solution: Dilute the stock solution with a suitable buffer (such as serum- and phenol red-free culture medium or PBS) to prepare a working solution with a concentration of 1-10μM. Please adjust the concentration of the working fluid according to the actual situation, and prepare it now to avoid repeated freezing and thawing. Notice: The working solution must be prepared fresh on the day of the experiment. Before use, Calcein Blue AM solution needs to be rewarmed and centrifuged.
2. Cell suspension staining
(1) Suspension cells: centrifuge at 4°C and 1000g for 3-5 minutes, discard the supernatant, and wash twice with PBS or other buffer, 5 minutes each time.
(2) Add the Calcein Blue AM working solution, incubate at room temperature in the dark for a short period of time (30 minutes), and then wash twice with PBS.
(3) Select the appropriate instrument and conduct fluorescence measurement.
3. Cell adhesion staining
(1) Culture adherent cells on sterile coverslips;
(2) Add the Calcein Blue AM working solution, incubate at room temperature in the dark for a short period of time (30 minutes), and then wash twice with PBS.
(3) Remove the coverslip from the culture medium, suck out the excess culture medium, and place the coverslip in a humid environment;
(4) Select the appropriate instrument and conduct fluorescence measurement.
4. Precautions:
(1) Fluorescent dyes all have quenching problems. Please avoid light as much as possible to slow down fluorescence quenching.
(2) For your safety and health, please wear a lab coat and disposable gloves when operating
(3) Calcein Blue AM should be stored in the dark throughout the experiment.
(4) This protocol and all reagents are for scientific research use only.
References:
[1] Grieshaber P, Lagrèze W A, Noack C, et al. Staining of fluorogold-prelabeled retinal ganglion cells with calcein-AM: A new method for assessing cell vitality[J]. Journal of neuroscience methods, 2010, 192(2): 233-239.
