Soybean Trypsin Inhibitor

Preparing a 0.05% Soybean Trypsin Inhibitor Solution:
Bring the Soybean Trypsin Inhibitor Solution to room temperature. Aseptically dilute the thawed SBTI 1:10 in D-PBS (e.g., 20 mL SBTI into 180 mL D-PBS). The final working concentration is 0.5 mg SBTI /mL. After dilution, 1 mL of SBTI working solution will inhibit 0.5 mg trypsin or 1 mL of 0.05% trypsin solution (specific activity is 10,000 BAEE units/mg protein).

Preparing a 0.1% Soybean Trypsin Inhibitor Solution:
Bring the Soybean Trypsin Inhibitor Solution to room temperature. Aseptically dilute the thawed SBTI 1:5 in D-PBS (e.g., 20 mL SBTI into 80 mL D-PBS). The final working concentration is 1.0 mg SBTI /mL. After dilution, 1 mL of SBTI working solution will inhibit 1.0 mg trypsin or 1 mL of 0.1% trypsin solution (specific activity is 10,000 BAEE units/mg protein).

Preparing a 0.25% Soybean Trypsin Inhibitor Solution:
Bring the Soybean Trypsin Inhibitor Solution to room temperature. Aseptically dilute the thawed SBTI 1:2 in D-PBS (e.g., 20 mL SBTI into 20 mL D-PBS). The final working concentration is 2.5 mg SBTI /mL. After dilution, 1 mL of SBTI working solution will inhibit 2.5 mg trypsin or 1 mL of 0.25% trypsin solution (specific activity is 10,000 BAEE units/mg protein).

1. Bring the following components to room temperature before use:
   1. D-PBS
   2. Trypsin or trypsin-EDTA
   3. Prepared Soybean Trypsin Inhibitor
2. Warm the complete growth medium to 37°C prior to use with the cells.
3. For each vessel, carefully aspirate the spent media without disturbing the monolayer. If the cell culture medium contains serum, each flask should be rinsed with D-PBS twice prior to adding trypsin or trypsin-EDTA.
4. Using 1 mL for every 25 cm², add the appropriate volume of trypsin or trypsin-EDTA to each vessel (e.g., each T-25 vessel would be dissociated with 1 mL trypsin or trypsin-EDTA).
5. Gently rock each flask to ensure complete coverage of the trypsin or trypsin-EDTA over the cells.
6. Observe the cells under the microscope. When the cells pull away from each other and round up (typically within about 3 to 6 minutes), remove the flask from the microscope and gently tap the culture vessel from several sides to promote detachment of the cells from the flask. Do not over-trypsinize as this will damage the cells.
   1. Some strongly adherent cell types, such as keratinocytes, may take much longer and may require trypsinization at 37°C.
   2. Some cell types may require more vigorous tapping.
7. When the majority of cells appear to have detached, quickly add 1 to 2 mL diluted Soybean Trypsin Inhibitor to each vessel for every for every 25 cm² of surface area. Gently pipette or swirl the culture to ensure all of the trypsin or trypsin-EDTA has been neutralized.
8. Transfer the dissociated cells to a sterile centrifuge tube and set aside while processing any remaining cells in the culture vessel.
   
   OPTIONAL RINSE STEP:
   When needed, add 3 to 5 mL D-PBS to the tissue culture vessel to collect any additional cells that might have been left behind. Transfer the cells and D-PBS to the centrifuge tube containing the dissociated cells. Repeat this rinse step as needed until all cells have been collected.
   
   
9. Centrifuge the cells at 125 x g for 5 to 10 minutes.
10. Aspirate neutralized dissociation solution and resuspend the cell pellet in 2 to 8 mL fresh, pre-warmed, complete growth medium.
11. Count the cells and seed new culture vessels at the recommended density.
12. Place newly seeded vessels in a 37°C, 5% CO₂, incubator, and incubate for at least 24 to 48 hours before processing the cells further.

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