Calcein Viability Assays Troubleshooting

As with all experimental techniques, calcein AM viability assays come with their own unique set of issues. Below are a number of issues a researcher could run into, and possible troubleshooting techniques.

Table of common difficulties, along with probable causes and solutions.

Issue	Possible Reason	Possible Solution
Low fluorescence	Too low of a concentration of calcein AM was used.	Increase the concentration of calcein AM used.
	The plate was not compatible with the microplate reader.	Black, solid-well plates should be used.
	Cells are not healthy.	A secondary assay assessing cell health can be performed to verify the health of the cells. Generally, >95% cell viability is acceptable.
	Plate was over exposed to light.	Ensure the plate is covered with aluminum foil after adding the working reagent, and then uncovered immediately before reading.
	Cells have lifted off the plate surface due to the use of strong reagents.	Use a microscope with a bright field light to check that cells are still present on the plate.
Poor duplicates or triplicates	Bubbles are present in the wells	Ensure careful pipetting. If bubbles are trapped, carefully centrifuge the plate at 4 ℃.
	Cells are not pipetted accurately.	Ensure proper resuspension of cells before pipetting, and that the pipette is calibrated. Before adding cells to wells ensure they have been appropriately mixed to obtain a uniform cell suspension.
	Cells were lost during wash steps.	Wash and aspirate cells gently. Do not disturb the cell pellet during aspiration.
	Only a small number of cells were available.	Cell loss could be reduced by leaving a bit of supernatant in the tube instead of removing all.
	Large variation in fluorescence intensity between repeats.	Usually due to differences in cell density across the plate and/or inhomogeneous distribution of cells within wells. Cells should be seeded at equal density across the plate.
High background fluorescence	Calcein AM working reagents are not fresh or close to expiration.	Prepare calcein AM working solution immediately before use. Use freshly diluted calcein AM. Read manufacturer information as some reagents may be susceptible to hydrolysis under moisture.
	Cells are not washed appropriately	Slightly increase the duration of the step. Ensure the use of the appropriate wash buffer.
	Cell culture media is interfering.	Phenol red used in media may interfere if analysis is performed under a fluorescent microscope.
	In some cell types calcein may sequester in intracellular organelles leading to artifacts.	Observe calcein-loaded cells under a fluorescence microscope to verify.
Over fluorescence	The density of the cells is too high	Add a preliminary step to measure cell count to ensure the appropriate number of cells are used. Ensure a well-pipetted cell suspension is used.
	Incubation time is too long.	Shorten the incubation time after calcein addition.

References

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Calcein Fluorescence Quenching to Measure Plasma Membrane Water Flux in Live Mammalian Cells
In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract
Protocol for Single-Cell Analysis of Tumor-Infiltrating B Cells Isolated from Human Breast Cancer Tissue Before and After Neo-adjuvant Chemotherapy
Protocol for Single-Cell Analysis of Tumor-Infiltrating B Cells Isolated from Human Breast Cancer Tissue Before and After Neo-adjuvant Chemotherapy