Obtaining Hemocytes and Hemolymph

Pelleting Hemocytes

1. Chill AC-saline. You will need 1-2 ml AC per larva.
2. Chill the larva 3-5 minutes under ice to give you a still insect and a good bleed. Also, keep the scissors on ice.
3. Bleed by cutting a proleg (or by inserting a needle tip into the dorsal blood vessel, except this we usually only resort to when we need really 'clean' samples because the yield will be less than cutting a proleg) and letting the hemolymph drip freely into the AC-saline in a polypropylene tube.
4. Mix the AC-saline and hemolymph well using the transfer pipettes. (Use a fresh pipette for each hemolymph sample; discard after use. We do this because sometimes mixing hemolymph from different animals will cause the cells to clump. Pooling hemocytes after hemolymph has been diluted away is generally fine.)
5. Pellet the cells by centrifuging at approximately 120 g for 10 minutes at 4C. Use 730 rpm on the Beckman Allegra 6R Centrifuge; higher than that and the cells will start to lyse.
6. Remove and discard (if we want the supe, too, see below) the supernatant, being careful not to disrupt the cell pellet.
7. Resuspend the cell pellet in 500-1000 µl of AC-saline. You can pool pellets at this stage so you will have pellets from 6-8 larvae in the same 1.5 ml tube. Label the tube with number of larvae, date, and, if they were injected note how many hours after injection they were bled (for purifying MS39 we want to bleed 36-40 hours after injecting.).
8. Centrifuge again, 120 g 10 min 4C.
9. Remove supernatant and put centrifuge tube in -80C freezer in suitable (injected or non-injected) box labeled hemocyte pellets.

• Make sure you wash the scissors very well, then spray them with 70 % ethanol.
  
• For notes on AC saline see Manduca Buffers.
  
• Make sure you use polypropylene tubes.
  
• In order to start to purify a protein, we will need pellets from approximately 400 Manduca.