Trypan Blue Staining Protocol

Background

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Trypan blue staining is a widely used technique in cell biology and microbiology to assess cell viability and count. It involves the use of Trypan blue dye, which selectively stains dead or damaged cells with compromised cell membranes. Viable cells possess intact membranes and exclude the dye, appearing unstained under a microscope, while non-viable cells take up the dye, causing them to appear blue. This method is commonly employed in cell culture experiments, cell counting procedures such as hemocytometry, and in assessing cell viability following treatments or experimental manipulations.

Protocol

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1. Prepare a 0.4% (w/v) trypan blue solution in PBS (phosphate buffered saline). A trypan blue solution can also be aliquoted and kept at -20°C for up to a year.
2. Determine the cell density in your cell suspension according to your laboratory’s standard protocol.
3. Dilute the cells as required by your method of counting (hemocytometer or cell counter).
4. Add one volume of trypan blue solution to one volume of the cell suspension. For example, add 100 µl of 0.4% trypan blue solution to 100 µl of cell suspension.
5. Immediately count the cells using a cell counter or hemocytometer. Determine both the number of blue cells (dead cells) and the total number of cells.
6. Calculate cell viability: % cell viability = (total number of cells - number of dead cells) / total number of cells x 100
7. A healthy cell culture typically shows ≥95% cell viability.

Tips and Tricks

1. If sterile, trypan blue solution can be kept at room temperature for several months, however, it will form aggregates and crystals over time, so it is ideal to either prepare fresh solutions or filter the solution with a 0.2-micron filter prior to use.
2. Maintain consistent staining time: Ensure that cells are stained for the same duration to maintain consistency across samples. Typically, a staining time of 3-5 minutes is sufficient, but this may vary depending on the cell type and protocol.
3. Handle cells gently: Avoid vigorous pipetting or agitation during the staining process to prevent mechanical damage to the cells, which could lead to false-positive staining.
4. Count cells promptly: Once stained, count the cells promptly to minimize dye diffusion and ensure accurate assessment of cell viability. Cells may become more permeable to the dye over time, leading to increased background staining.
5. Use proper microscopy settings: When visualizing stained cells under a microscope, adjust the settings (e.g., brightness, contrast) to enhance the visibility of stained cells while minimizing background noise.
6. Clean equipment thoroughly: make sure to clean the hemocytometer and use autoclaved or sterile-packaged pipette tips and plasticware to prevent contamination and ensure accurate staining results.