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Cellular and humoral responses in animals are frequently induced by subcutaneous injection with antigen emulsified in an agent such as complete Freund’s adjuvant (CFA). These emulsions are commonly prepared by repetitive mixing of aqueous and oil components in two coupled syringes (1). Although effective, this procedure is slow and tedious. Sonification has proven to be a more rapid alternative, but loading the emulsion into syringes is awkward and wasteful, particularly for small volumes. We have devised a simple modification to the sonication method of adjuvant emulsification that improves the loading of syringes and markedly reduces the amount of emulsion lost during this loading procedure. In preparation for emulsification, equal volumes of adjuvant and aqueous antigen solution are added to a sterile polypropylene tube (No. 2059 Falcon 14-mL tubes, 17 × 100 mm; Becton Dickinson Labware, Lincoln Park, NJ, USA). Volumes of 0.5 to 8.0 mL could be safely emulsified in these tubes. The sample was emulsified with a Branson Sonifier (Branson Ultrasonic, Danbury, CT, USA) on an output control setting of 3–4 and duty cycle of 75% for approximately 3–10 seconds. The emulsification is judged complete when the sample tube can be inverted without movement of the emulsion. Formerly, emulsion prepared in this manner was drawn into syringes for injection; but, in practice, the emulsion has proven difficult to draw up and is prone to airbubble formation, which compromises animal injections. Furthermore, a considerable proportion of the emulsion is often lost during the loading of this sonicated emulsion into syringes. The inefficiency of loading emulsion prepared in this manner has been particularly troubling when small volumes of limited or expensive antigens were involved. To take advantage of the speed of sonication for emulsion preparation while reducing waste, we have devised the transfer technique diagrammed in Figure 1. To load syringes, the bottom of the polypropylene tube containing the emulsion was pierced with an 18gauge sterile needle, and the tube was placed over the top of an empty glass syringe. The plunger of a 10-cc disposable syringe (Luer Lok No. 309604; Becton Dickinson, Franklin Lakes, NJ, USA) was placed into the polypropylene tube containing the adjuvant and firmly pressed to the bottom of the tube. The plunger fit loosely at the top of the polypropylene tube, but formed a tight seal near the base because of the tapered sides of the tube. Moderate pressure was sufficient to expel virtually all of the emulsion. An emulsion volume of 0.5 mL was the lower practical limit for this technique. From a starting volume of 0.5 mL (comprised of 0.25 mL CFA and 0.25 mL aqueous antigen), 80.8% of the emulsion was recovered from the tube. A recovery of greater than 95% was achieved with emulsion volumes of 2.0 mL or greater. Therefore, this method permits rapid emulsification with minimal waste, permitting a more conservative formulation of emulsions, which can be particularly valuable when the quantity of available antigen is limited. The practical upper limit of this technique is approximately 8 mL. For greater volumes, CFA and antigens can be mixed in larger tubes and divided into several aliquots for emulsification. To test the efficacy of this method, we compared the T-cell response in BALB/c mice immunized with the chicken ovalbumin peptide 323-339 (OVA 323-339), emulsified in complete Freund’s adjuvant by the traditional dual-syringe technique and by sonification. Ten days after immunization, draining popliteal lymph nodes were removed and single-cell suspensions

Transfer Technique for Minimizing Waste of Sonified Adjuvant Emulsions