CELL COUNTING PROTOCOL Time Period – 10 min Formulation of Trypan Blue Solution 1. Obtain 100 ml bottle of trypan blue powder (StemCell Technologies Inc.) 2. Trypan Blue - Mix g of trypan blue with 100 ml of Phosphate Buffering Solution (PBS). | CELL COUNTING PROTOCOL Time Period - 10 min Formulation of Trypan Blue Solution 1. Obtain 100 ml bottle of trypan blue powder StemCell Technologies Inc. 2. Trypan Blue - Mix g of trypan blue with 100 ml of Phosphate Buffering Solution PBS . 3. Will remain stable for two years while stored at room temperature. Viable Cell Counting 1. Dilute Cells l lin Trypan Blue. Add lOplof trypan blue to a microcentrifuge tube. Using sterile pipette tips and pipette aid add alOplof the sample taken from the center of the 15 ml conical tube to the trypan blue. Sterilize the pipette with alcohol and let dry before taking aliquot from sample. Make sure that that the sample comes in contact with the dye. Allow the solution to sit for 5-15 min. Trypan blue stains non-viable dead cells. Be sure to thoroughly mix Trypan Blue with sample in microcentrifuge tube using pipette. 2. Hemocytometer preparation. To clean rinse the chamber with distilled water and then alcohol and air dry. Position the cover slip over the chambers. 3. Place lOplof the mixture in both wells of the hemocytometer. Todo this center the cover slip on the hemocytometer hold the cover slip at its sides with two fingers lay the pipette tip as parallel to the ground as possible and inject the mixture in the well underneath the cover slip. Make sure the entire surface of the rectangular grid of the hemocytometer is covered. It is important not to under fill or overfill the chambers. 4. Count viable cells using 100x that overlay the grid of the hemocytometer at the corners and at its center. If cells are on the border outlining each square count only the cells on the top and left border of the square as shown below. 5. Count both sides of the hemocytometer using the hand counter while looking through the microscope. Count 5 of the 10 sub squares on each side of the hemocytometer. See example below. 6. Use the following calculations to determine the number of cells per milliliter. 135 I l l l Hemocytometer Chamber .
