Thawing Frozen Serum

Purpose
The following protocol is suggested thaw frozen serum. Frozen serum must be mixed after complete thaw to ensure uniformity.

Equipment
Refrigerator capable of maintaining 2 to 8°C, optional

Materials
Frozen Bovine Serum

Specifications
Small amounts of precipitates may form after thaw procedure and are not uncommon, even in serum that is thawed using proper technique. The precipitates are not toxic to cell cultures, but will affect the appearance and consistency of each bottle of serum.

Precautions

• Multiple thaw/freeze cycles should be avoided.
• If thawed serum is allowed to stand for long periods of time, greater amounts of precipitates will form and are often insoluble.
• If serum is not periodically agitated during thaw process then a gradient will form throughout the liquid portion in the bottle going from light to dark. This is the result of lipids, proteins and salts settling out and concentrating at the bottom of the bottle.
• Filtering serum to remove precipitates is not recommended and could result in loss of nutrients.
• If improperly thawed serum can congeal, resulting in a jelly like substance that will not re-suspend and may not be suitable for use.

Procedure

1. Thawing at 2-8°C
   1. Place frozen serum in refrigerator capable of maintaining 2 to 8°C.
   2. Check bottles periodically for complete thaw.
2. Thawing at 37°C
   1. Fill the water-bath with sufficient water so that the product is immersed near the serum level. Do not immerse bottles passed the threaded cap.
   2. Place each bottle in 37° water bath.
   3. Gently swirl or shake the bottles every 10 minutes until the serum is completely thawed, or use a shaking water bath to agitate the serum while thawing.
3. Swirl bottles after complete thaw to create uniformity.
4. Serum should be stored in useful aliquot volumes typical for application and refrozen for future use.