Dead cell counts during serum cultivation are underestimated by the fluorescent live/dead assay

Area of Science:

• Cell Biology
• Biotechnology

Background:

• Live/dead fluorescent assays are common for assessing cell viability.
• These assays often use DNA-binding dyes like propidium iodide (PI) to detect dead cells with compromised membranes.

Purpose of the Study:

• To investigate the impact of serum in cell culture medium on the accuracy of live/dead fluorescent assays.
• To determine if serum affects the detection of dead cells by DNA-binding dyes.

Main Methods:

• Chondrocyte viability was assessed using a live/dead assay kit and trypan blue staining.
• Cultures were maintained with and without serum, and with a DNase I inhibitor in serum-containing medium.

Main Results:

• Propidium iodide (PI) failed to detect dead cells in serum-containing medium after 2 days of culture.
• Dead cells were detectable by PI in serum-free medium or when DNase I was inhibited.
• Trypan blue staining consistently detected dead cells across all conditions.

Conclusions:

• Serum contains DNase I, which degrades DNA in dead cells, rendering them undetectable by DNA-binding dyes.
• The presence of serum can compromise the reliability of live/dead fluorescent assays for cell viability assessment, especially after prolonged culture.