FBS heat inactivation

  • Heat Inactivation (HI): a process by which serum is maintained at a temperature of 56 ± 2°C for 30 ± 2 minutes.

Note

You should use a control bottles to measure temperature. The control bottle should be equivalent to the product bottle. Controls bottles are volumed to the same level as the product being heat inactivated and fitted with a thermometer suitable for monitoring 56°C. The thermometer should not touch the sides or bottom of the bottle. The temperature of the control must be 56± 2°C for 30 ± 2 minutes during the heat inactivation process. A 15 mL conical works well to heat-inactivate a 10 mL sample (good for 100 mL of DMEM/FBS)

  1. Thaw serum. If serum was thawed in a refrigerator allow serum to come to room temperature prior to placing in water bath.

  2. Fill the water-bath with sufficient water so that the product and control bottles are immersed near the serum level.

  3. Set water-bath temperature to maintain the product at 56± 2°C.

  4. Place a lead weight on the control bottle and place into the center of the water-bath.

  5. Mix the contents of the product bottles using a gentle swirling motion until the product is uniform.

  6. Place lead circular weights over the tops of the bottles to keep them upright.

  7. Place the bottles in the water-bath.

  8. Swirl bottle thoroughly every 10 minutes or if applicable turn on the oscillating shaker unit. Check the temperature of the control bottle frequently as the temperature approaches 56±2°C.

  9. When the temperature of the control reaches 56± 2°C start the timer for 30 minutes. If a shaking water bath is not available, ensure bottles are swirled every 10 minutes during the entire process.

  10. After 30 minutes turn off the oscillating shaker and remove the bottles from the water bath. Cool to room temperature and either aliquot or return to the freezer.

Note

Denatured protein resembles a jelly like substance, usually at the bottom of the bottle of serum. If this occurs, decant serum and use. Denatured protein should not be confused with Fibrin, a fine precipitate sometimes seen in thawed serum. Fibrin precipitate is a fine whitish powder flake, which has no ill effects on cell culture.