Using Trypan Blue to Check Cell Viability

Overview

When preparing cells for an experiment, it is useful to know what percentage of them are alive. This protocol will walk you through the process of checking cell viability during cell counting by using trypan blue. 

Materials

• Cell Suspension
• Cell Culture Medium
• Trypan Blue (0.4% w/v solution)
• Microcentrifuge Tubes
• Micropipettes
• Micropipette Tips
• Microscope
• Hemocytometer
• Cover slips
• Cell counter

Methods: Trypan Blue

1. After preparing your suspension in 1-5 mL of culture medium (as done on step 2 of the Hemocytometer Cell Counting protocol), pipette 20 µL of cell solution into a microcentrifuge tube;
2. Add 20 µL of trypan blue;
   1. In this 1:1 proportion, the dilution factor for hemocytometer cell calculations is 2. You may also mix trypan blue with your cell solution in other proportions, but remember to appropriately change the dilution factor for your calculations.
3. Pipette up and down to thoroughly mix the two solutions;
4. Pipette 10 µL of the resulting solution in the hemocytometer chamber;
5. Carefully place a cover slip on top, ensuring that no bubbles are trapped in the system;
6. Place hemocytometer under the microscope;
7. Using the differential cell counter, count normal cells on one side, and blue cells on the other side. Dead cells are blue because trypan blue can only permeate damaged cell membranes; 
8. You can calculate cell viability by using the following formula: 100*(live cells)/(dead cells + live cells).