Http://www.jove.com/video/4173/a-simple-protocol-for-extracting-hemocytes-from-wild-caterpillars

I have had no problems with extracting "blood" from honeybees, polistes, etc. You should not inject anything, or your sample will be a mixture of your saline and real hemolymph. Just poke a tiny hole between the intersegmental membrane and use a capillary tube to draw them out. Photos for bees are here: http://cyberbee.net/gallery/index.php/research/bleeding

yes dear Antonino

It depends upon howmuch haemolymph is required. If very small amount is required it can be obtaining by cutting the appendages and the oozing liquid can be taken on slides. In case your requirement is more as mentioned by Dr.Huang, from the inersegmental region it can be drawn by making a puncture and using a capillary tube.

I am sorry, I do not konow. Try to contact Dr. Rettich in Czech republic, [email protected]

Cut antennae haemolymph may ooze out

You can get it from intersegmental region by inserting 27 number needle.

It is not clear why you are injecting saline befor collecting the hemolymph. I have not experienced much difficulty in collecting large drops from a cute leg or antenna, while applying gentle pressure to the abdomen. Most insects maintain a slightly negative pressure in the hemocoel

If you want larger amounts, seal the mouth and anus with Tackiwax, open a wound in the frons and/or neck and centrifuge, using a tube of appropriate size and shape to keep the insect above the bottom of the tube. The tube should be sinalised or silicane treated to keep the fluid from sticking

It is not clear why you are injecting saline before collecting the haemolymph. It could be mix of haemolymph with saline. I have experienced not difficulty in collecting few drops of haemolymph from tick legs for heamolymph test or test Gimenez. (Gimenez 1964, Burgdorfer 1970).

I wouldn't recommend injecting saline either, your results from such samples might be artifacts of poor experiment design. You could try the following: depending on the size of your species, you could use a centrifuge to collect haemolimph, we used to do it to collect haemolimph from mosquitoes. Puncture a 250ul epp a few times in the bottom. Fit 250ul epp into 1.5 ml epp. Take tour specimen, puncture a few holes in posterior abdomen (sometimes cutting a bit of the exoskeleton is better, trying to avoid internal organs IMPORTANT, otherwise you will collect undesired fluids. Place specimen inside epp with punctures/cut towards the bottom. Centrifuge at 1000G or increase G to improve. If your cockroach is huge, try using punctured 1.5 epps and fit into 15 ml falcons and centrifuge. If it NEEDS to be from buccal cavity, try cutting off some of it and put the insect facing down tube and perform centrifuge extraction.

To extracted haemolymph add your content

From buccal cavity -with forceps pull mouth parts haemolymph will ooze out.

The volume of haemolymph is relatively small to what most people think. The best way to collect haemolymph is by using very small capillary tubes 1-5 micro liter tubes

Stretch head ,for neck to eject and make half cut at the neck for haemolymph to ooze out.

Same as you, I did not find these traditional methods useful. I am studying immune responses in the american cockroach so extract hemolymph all the time. We essentially use a modification of the protocol for mosquitoes (Video Article

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito

Aedes aegypti. Amina A. Qayum, Aparna Telang. Department of Biology, University of Richmond). What we do is inject 80uL of buffer that is 60% Schneider's insect medium, 10% Fetal Bovine Serum and 20% Citrate Anticoagulant buffer (anticoagulant; 98 mM NaOH, 186 mM NaCl, 1.7 mM EDTA, 41 mM citric acid, pH 4.5). We use an insulin syringe to do this into an anesthetized cockroach (we use CO2 to knock out). We then leave the cockroach alone for 10 minutes. We then perfuse the cockroach with 250uL of Schneider's medium + 10% FBS using a BIG needle (like 27 guage) in the abdomin and while that perfusion is occuring a second person collects the effluent (from the injection site) with a 100uL micropipette. We get about 2.5x10^5 live cells per cockroach this way. I would love to talk to you about what you are examining in case we can collaborate! Let me know.