Cellular Viability--Calcein / Propidium Iodide

We’ve performed this assay on the following cell types:

• Dissociated Primary Cortical Neurons Plated at 0.2 - 1.0x106 cells/ml (100 μl/well of 96
well plate; 1 ml/well of a 24 well plate; 4 ml/well of 6 well plate).
• Dissociated Primary Hypothalamic Neurons Plated at 0.4 – 0.6x106 cells/ml

a. Calcein and Propidium Iodide Assay Protocol:

• The calcein assay is based on the conversion of the cell permeant non-fluorescnt calcein
AM dye to the fluorescent calcein dye by intracellular esterase activity in live cells.
• Propidium iodide (PI) is membrane impermeant and therefore does not enter viable cells
with intact membranes. When PI does gain access to nucleic acids and intercalates its
fluorescence increases dramatically and is therefore used to identify dead cells.
• Calcein can propidium iodide (PI) can be use separately or together to assess cellular
viability or cell death, respectively.

Assay
• Following drug/compound treatment @ 37° C remove the media and wash the cells.
• Serum contains esterase activity that will greatly increase the background signal
therefore at least remove the media and if possible wash the cells once.
• Remove media from each well using a blue pipette tip being careful as not to remove
cells.
• Gently wash cells once with 200 μl (for 96 well plate) or 400 μl (for a 24 well plate) warm
(37°C) 1X DPBS (containing Mg and CaCl2).
• To avoid removing loosely attached cells don’t wash if the background signal is not too
high
• If using PI - don’t wash as the dead cells/DNA will be lost.
o As controls:
ƒ Wells containing no cells + calcein dye and/or PI to determine the level of
the background signal.
ƒ Wells containing cells and treated with 0.2% Triton X-100 (10-15 min
treatment) to determine the background signal from dead cells for both
calcein and PI assays.
• Add 200 μl (for 96 well plate) or 400 μl (for a 24 well plate) per well of 3 μM calcein AM
(live dye) or 2.5-5.0 μM PI (dead dye) diluted in warm (37° C) 1X DPBS.
o Calcein AM stock = 1.005 mM (1 mg/ml; aliquots are stored @ -20°C)
o PI stock = 1.5 mM (1 mg/ml; stored @ @ -20°C).
• Incubate for 30 min @ 37° C (when taking pictures it is not necessary to wait this long).
• Detect absorbance from the top of the plate using a plate reader:
o For calcein detect absorbance @ 485 nm/535 nm.
o For PI detect absorbance @ 530 nm/620 nm.
• To determine the background from un-metabolized/incorporated/PI:
o Carefully remove the dye using a multiple channel pipette
o Add 200 or 400 μl of warm 1X DPBS
o Read again @ 485 nm/535 nm and/or 530 nm/620 nm.

Ordering information:
• Calcein AM, Molecular Probes/Invitrogen #C3099, 1 ml.
• Propidium Iodide (1 mg/ml in water), Molecular Probes #P-3566.