The Trypan Blue Exclusion Test is a method to assess cell viability using light microscopy. Live cells have intact membranes that exclude trypan blue, resulting in clear cytoplasm, while dead cells take up the dye, showing blue cytoplasm. The protocol involves suspending cells in PBS with trypan blue, mixing, and examining under a microscope. Cells are counted in a hemacytometer, and viability is calculated based on the ratio of unstained (viable) to stained (nonviable) cells. The test requires a 0.4% trypan blue solution and involves centrifugation, resuspension, mixing, and counting. The viability percentage is calculated by multiplying the number of viable cells by 2 (due to dilution). The test is simple and rapid but may have limitations due to subjective assessment of dye uptake. Flow cytometry offers more precise measurements but is less suitable for complex cell purification techniques. Recent automated fluorescence microscopes can assess cell viability more rapidly and accurately. Other methods, such as DNA-binding dyes and Annexin V, also assess viability but often require flow cytometry. The Trypan Blue Exclusion Test can be completed in 5–10 minutes. Key references include Shapiro (1988) and Avelar-Freitas et al. (2014).The Trypan Blue Exclusion Test is a method to assess cell viability using light microscopy. Live cells have intact membranes that exclude trypan blue, resulting in clear cytoplasm, while dead cells take up the dye, showing blue cytoplasm. The protocol involves suspending cells in PBS with trypan blue, mixing, and examining under a microscope. Cells are counted in a hemacytometer, and viability is calculated based on the ratio of unstained (viable) to stained (nonviable) cells. The test requires a 0.4% trypan blue solution and involves centrifugation, resuspension, mixing, and counting. The viability percentage is calculated by multiplying the number of viable cells by 2 (due to dilution). The test is simple and rapid but may have limitations due to subjective assessment of dye uptake. Flow cytometry offers more precise measurements but is less suitable for complex cell purification techniques. Recent automated fluorescence microscopes can assess cell viability more rapidly and accurately. Other methods, such as DNA-binding dyes and Annexin V, also assess viability but often require flow cytometry. The Trypan Blue Exclusion Test can be completed in 5–10 minutes. Key references include Shapiro (1988) and Avelar-Freitas et al. (2014).