Obtaining Hemocytes and Hemolymphs

Immune-Activating Manduca

• Make a 1 mg/ml solution in PBS. If making 100 ml, weigh 100 mg. pH to 6.8 with KOH or HCl as needed.
• Weigh this carefully. The bacteria should be dead but don't trust that too much and be careful and clear up after you.
• Autoclave (liquid cycle in small autoclave upstairs for twenty minutes), then let cool slowly.
• In sterile hood, aliquot into 1 ml amounts and store -20C in tubes labeled M.l. in box that is well labeled with how you made the solutions and the date.

• Chill larvae briefly on ice, if desired. They will not wriggle so much then.
  
  
• Use 30 gauge syringe needle with 1 ml barrel and inject between 50-100 µl of the M. lysodeikticus solution into the abdominal segment that contains the second set of prolegs. Inject on the side of the larva, inserting the needle so the tip is just under the cuticle but about 1 cm forward from the insertion site. (i.e., the needle is parallel to the long axis of the larva, and inserted sufficiently so if the larva wriggle they will not cause the M. lysodeikticus to ooze out.)