Hemoglobin, ≤20 mg/dL
cell culture | mammalian: suitable
≤10 EU/mL Endotoxin
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Heat inactivation of serum is performed to inactivate complement (which can cause cell lysis or downstream problems in immunoassays). If there are immunoglobulins present in the serum that can bind to the cells in culture (either specifically or non-specifically), there is a chance for antibody-mediated complement cytotoxicity (ADCC, causing cell death). It may also reduce the titer of some mycoplasma and some viruses. Often, heat-inactivated serum is used because of the adoption of a previous protocol. It is mainly used when culturing hematopoetic cells or performing immunological type assays such as mixed-lymphocyte reactions or neutralization assays.FBS may not be heat inactivated as this is not required for all applications. It limits the loss of the other protein activity that may be minimally present in the serum.A protocol for heat inactivation:1. Allow serum containers to come to room temperature. Swirl bottles of serum immediately before adding to the water bath.2. A water-filled control bottles (T1) should be placed in a 56 °C water bath. Place the containers in the water bath up to the serum line. Do not completely submerge the containers. When the temperature of T1 reaches 56 °C, start the timer set for 30 minutes. Do not allow temperatures to exceed 56 °C. Higher temperatures will denature the proteins, gel the serum or alter the performance of the serum.3. Gently swirl the bottles every 5 to 10 minutes while in the water bath and check the temperature in the control bottle. It is not uncommon for plastic containers to bow slightly after heat treatment. This is not harmful to the serum.4. At the end of 30 minutes remove the serum bottles from the water bath. Aliquot the serum into smaller volumes and freeze. Sigma-Aldrich does not recommend repeated thaw-freeze cycles.
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