Swirl the bottles every minutes to ensure uniform heating of the serum. The serum should be left overnight before aliquotting. Heat-inactivated serum may be re-frozen once, thawed and then used. The serum should be stored in small aliquots that can be thawed individually as needed. We do not recommend more than one freeze-thaw cycle after heat-inactivation. Remember me. Request new password. Commands Support portal Log in. These aggregates would produce false-positive tests for immune complexes and could inhibit a variety of cell-mediated reactions in assays which incorporate heat-inactivated serum.
Other temperatures were tested to determine whether endogenous haemolytic activity could be destroyed without forming immunoglobulin aggregates. At 53 degrees both endogenous haemolytic activity and heat-labile ACA were inactivated and formation of heat-stable ACA in normal serum was minimal.
To understand the structural involvement responsible for mediating the corona composition, serum was heat inactivated and native clusterin was re-added to the protein mixture. We found that native clusterin, which was re-added to heat inactivated serum, adsorbed to PS-PEG NC clusterin band marked with a red star.
This proves that in a complex protein mixture it is the protein structure that critically determines the adsorption behavior, which eventually affects the cellular outcome.
To further reveal the influence of the protein structure on corona formation, additional experiments were performed using a broad range of different nanocarrier systems. All nanocarriers were incubated with native or heat inactivated serum and plasma. Proteomic analysis highlighted the major differences in the corona composition for HES nanocapsules incubated with the respective protein source Fig.
As shown above, the adsorption of clusterin was prevented when HES nanocapsules were incubated with heat inactivated serum or plasma. In addition, we found major differences in the corona pattern for serum compared to plasma Fig. As already reported in the literature, the protein source can influence corona formation. This can be explained by the fact that complement proteins require calcium to maintain their native structure and function.
Additionally, cellular uptake studies of HES nanocapsules coated with serum or plasma native vs. Serum incubation strongly enhanced cellular uptake, which is mainly attributed to the involvement of complement proteins. Due to heat inactivation of serum or plasma the overall amount of complement proteins was decreased strongly, which resulted in decreased cellular interaction.
This can probably be attributed to the lower amount of clusterin in the corona after heat inactivation, which is comparable to the results presented for PS-PEG NC.
DOI: Received 12th September , Accepted 1st November Abstract Adsorption of blood proteins to the surface of nanocarriers is known to be the critical factor influencing cellular interactions and eventually determining the successful application of nanocarriers as drug carriers in vivo.
A The macrophage cell line RAW C The most abundant proteins TOP 25 are summarized in the heat map highlighting the major protein variations. The clusterin band marked with a red star appears under reducing conditions at a molecular weight of 38 kDa. Differential scanning fluorimetry nanoDSF was used to monitor protein folding upon heating.
A Schematic illustration of the unfolding process and exposure of tryptophan residues here shown for human serum albumin PDB structure 1AO6. B Unfolding and refolding curves of clusterin in PBS 0. The fluorescence intensity at nm and the corresponding first derivative are shown.
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