Preparing Manduca sexta Hemocyte Slides

• Avoid polystyrene, use polypropylene tubes.
  
• Do not let the formaldehyde fixation go too long. Ten minutes is sufficient.
  
• Speed of centrifuge is important. At too high g-forces, the cells will lyse.
  
• Keeping cells cold is important. Make sure solutions and centrifuge are prechilled and that you work as quickly as reasonably possible. Plan to avoid interruptions.
  
• When making slides comparing bacteria-challenged to non-challenged, look for an initial (1 minute) lysozyme assay difference (absorbance change at 450 nm, over 1 minutes, use 20 ƒl of hemolymph) of at least 2.5 times higher for the bacteria-challenged compared to the controls. See Lysozyme Assay under Techniques: Assay.
• CAUTION: Hazardous formaldehyde gas is evolved as paraformaldehyde dissolves. NEVER microwave formaldehyde!
  To make 5% formaldehyde: add 10ml Manduca saline buffer no Ca²⁺ Mg²⁺ to 0.5g paraformaldehyde. Dissolve by heating in 55-60C water bath. If the solution remains cloudy, add 1-2 drops of 1M KOH. The obtained formaldehyde solution may be suitable for use for ~2 weeks if refrigerated.
• TTBS is TBS with Tween 20 to 0.05%. To make 50 ml TTBS add 2.5mls of 1% Tween. (To make the 1% Tween, add 100 µl of 100% Tween 20 to 10ml TBS. Hint: cut off tip of pipet tip before attempting to pipet the 100 µl; pipet very slowly; to disperse position the tip below the surface of the TBS and again pipet very slowly, then slowly suck up and disperse several times into the TBS.)
• Blocking solution: 2% BSA in TTBS: to 50ml TTBS add 1g BSA and 0.5ml 100x thimerosol (as a preservative).

1. Chill fifth instar larvae on ice for 3-10 minutes.
2. Bleed by cutting proleg (or by inserting tip of syringe needle into dorsal blood vessle if procedure calls for it). Collect only the hemolymph that drips freely into 1-2 ml AC saline on ice (if using syringe needle, bleed into 500 µl AC in 1.5 ml microfuge tube), one larvae's hemolymph per tube. Try avoid having the piece of proleg drop into the solution: if it does, remove immediately after you bleed, otherwise the hemocytes will attach to it.
3. Pellet hemocytes by spinning 730 rpm in Beckman Allegra. Spin 10 minutes 4C. This corresponds to about 120 g.
4. Resuspend cells in AC-saline. You can pool cells from different animals at this stage, taking the solution from one tube into the next tube, etc.. So, cells from about 6 larvae can be pooled into a single 1.5 ml total volume of AC-saline in a microcentrifuge tube. Pellet again as above.
5. Resuspend cells in suitable amount of Manduca saline (containing calcium and magnesium). One larvae's worth will yield about 4 slides worth of cells. Resuspend so you can plate about 8 µl of cells per well on the slides. Slides have 12 wells each.
6. Apply to slides and let settle for 20 minutes.
7. Add equal volume of 5% formaldehyde fixative made from paraformaldehyde. CAUTION: remember formaldehyde is hazardous. Incubate the cells for 10 minutes with formaldehyde. This fixation time may be important for some antibodies. We have seen reduced staining intensity if fix has remained on for longer than 1 hour.
8. Remove the formaldehyde by careful aspiration into the bottle for formaldehyde waste, then rinse once with TBS (Manduca saline may give slightly better morphology, but that is not necessary.) Both the formaldehyde solution and this rinse need to go into the formaldehyde waste bottle.
9. Aspirate the TBS rinse into the formaldehyde waste bottle.
10. Add 1-2% BSA in T-TBS (or in Manduca saline plus 0.5% Tween 20) and incubate 1 hour. This blocks non-specific binding sites. If the slides were not for immuno-staining, then this step can possibly be eliminated. To make 10 ml of 2% BSA in Manduca saline plus 0.05% Tween, add 0.2g BSA and 0.5ml 1% Tween to 10ml MSB (the numbers are close enough).
11. Rinse once with TBS. Remove the TBS, leaving only a thin film on the wells, and promptly put the slides in a slide box and chill so that the wells are horizontal while freezing.