Cell viability assay by dye uptake assay

Summary

Living cells can take up diacetylfluorescein and hydrolyze it to fluorescein, which cannot pass through the cell membrane of living cells [Rotman and Papermaster, 1966]. Living cells emit green fluorescence, while dead cells do not. Cells that are not viable may also be stained with propidium iodide and subsequently emit red fluorescence. Cell viability can be expressed as the percentage of cells that fluoresce green. The method can be used for CCD analysis or flow cytometry (see Section 21.7.2). Source: Animal Cell Culture: A Guide to Basic Techniques, Fifth Edition

Operation method

Scheme 22.2 Dye uptake assay for cell viability assay

Principle

Cell suspensions were stained with a mixture of propidium iodide and diacetyl fluorescein and detected by fluorescence microscopy or flow cytometry.

Move

makings

non-sterile

single-cell suspension
Fluorescein diacetate, 10ug/ml, dissolved in HBSS
Propidium iodide, 500ug/ml

Non-sterile

Fluorescence microscope
Filter
Fluorescein: excitation 450/590 nm , emission LP 515 nm
Propidium iodide: excitation 488 nm, emission 615 nm

Procedure

1. The cell suspension is prepared in the same way as for the dye rejection method (see Scheme 22.1), but without phenol red in the growth solution.
2. Add the fluorescein dye mixture at a ratio of 1 : 10, with a final concentration of l ug/ml for diacetylfluorescein and 50ug/ml for propidium iodide. :.
3. Place the cells at 37°C and incubate for 10 min.
4. Place a drop of cells on a slide, cover with a coverslip and observe with a fluorescence microscope.

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