Live/Dead Stain for Human Foreskin Fibroblasts (HFFs)?

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Saif Wahid

One common staining method to assess cell viability is the use of fluorescent dyes such as propidium iodide (PI) and calcein-AM. Here's a general staining protocol you can consider using for live/dead staining of human foreskin fibroblasts (HFFs):

Materials:

Propidium iodide (PI)

Calcein-AM

Phosphate-buffered saline (PBS)

Fluorescence microscope

Procedure:

Prepare a working solution of PI by diluting it in PBS according to the manufacturer's instructions. Similarly, prepare a working solution of Calcein-AM.

Harvest the HFFs using a suitable method (e.g., trypsinization) and transfer the cell suspension to a sterile tube.

Centrifuge the cell suspension at a low speed (e.g., 200-300 × g) for a few minutes to pellet the cells.

Carefully remove the supernatant, ensuring not to disturb the cell pellet.

Resuspend the cell pellet in a small volume of PBS to create a cell suspension. The exact volume will depend on the cell density and the size of the culture vessel.

Take a small aliquot of the cell suspension and count the cells using a hemocytometer or an automated cell counter. Adjust the cell suspension volume with PBS to achieve a desired cell density for staining.

Transfer a portion of the cell suspension to a sterile microcentrifuge tube and centrifuge it at low speed for a few minutes to pellet the cells.

Resuspend the cell pellet in a small volume of the staining solution containing both PI and Calcein-AM. The final concentration of PI and Calcein-AM may vary depending on the manufacturer's instructions and the desired staining intensity.

Incubate the cell suspension with the staining solution for the recommended duration, usually 15-30 minutes at room temperature, protected from light.

After the incubation period, carefully remove the staining solution without disturbing the cell pellet.

Gently wash the stained cells with PBS to remove any excess dye and debris.

Resuspend the cells in a small volume of fresh PBS for examination under a fluorescence microscope.

Analyze the stained cells using appropriate fluorescence filters. Live cells will show green fluorescence due to the conversion of non-fluorescent calcein-AM to fluorescent calcein, while dead cells will exhibit red fluorescence due to the uptake of PI, which stains the nuclei of non-viable cells.

Malcolm Nobre

Hello Alexander Redmond

A method based on crystal violet staining may be used to evaluate Human Foreskin Fibroblasts cell viability. This cationic dye works as an intercalating agent that enables the quantification of DNA, which is proportional to the number of cells in culture, and the dye is easily sequestered by viable cells.

For more information you could refer to the Basic Protocol 2 in the article attached below.

Article Using Human Primary Foreskin Fibroblasts to Study Cellular D...

Best.