1. Clearly identify each animal by ear punch, for example, animal #1 will have no punch, #2 will have a single punch in the left ear, and so on.

2. To test whether the adjuvant is sufficiently emulsified, it can be transferred to a 27-gauge needle and expressed onto the surface of water. If it spreads and mixes with the water, it is not sufficiently mixed. Reconnect the syringe to the double-hub needle and mix for another min. If the emulsion is performed correctly, the mixture will remain separate from the water and maintain its shape.

3. Subcutaneously is between the skin and the underlying musculature. It is most accessible by tenting the skin first by pinching the skin with your fingers.

4. Intraperitonealy is in the abdomen. Scruff the mouse and flip it over so its belly is up. Inject off of the midline.

5. Intravenously is often done through the tail vein. This is a difficult procedure and best performed by a trained animal technician.

6. Place the plate in a drawer on a piece of white paper and check it every 2 min.

7. SP2/0Ag14 cells divide more than once every 24 h and must not be overgrown when used for fusions. They should be in logarithmic growth phase for use.

8. Cells are usually pelleted in a clinical centrifuge which may not have a 'g-force' conversion. Use 1000-1500 rpms.

9. Carefully hold the plate up to the light, looking up through the bottom of the wells. Take care not to tip the plate, as media on the lid is a vector for contamination to enter the wells.

10. Many investigators add feeder cells (i.e., peritoneal wash-out cells, or splenocytes) to produce conditioned media that seems to enhance hybridoma growth and cloning. However, the addition of cells can sometimes be a source of contamination, thus, as a source of conditioned media, we have harvested the supernatants from cultured splenocytes, filter-sterilized it, and used it to supplement the single-cell cloning experiments. The following is a protocol for obtaining peritoneal wash-out cells. Peritoneal cells are obtained from normal mice by flushing the peritoneal cavities with 3-4 mL of 0.34 M sucrose that has been filter-sterilized. The mouse is sacrificed, dipped in 70% ethanol, and laid on its back on toweling in a laminar flow hood. A snip is made in the skin between the hind legs and the skin is peeled forward. A pair of forceps is used to hold up the thin musculature over the abdomen and the sucrose can then be injected and withdrawn. Be careful not to puncture the gut. The peritoneal washes are pooled and centri-fuged and plated at approx 2 x 104 cells/well.

11. The amount of lysate will vary depending on how abundant the protein is and where in the cell it is located, for example, nuclear proteins usually require 3 times as much lysate.

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