Techniques

Technique figure

Note: This figure is intended to serve only as a guide to those new to the ecoimmunology field. We expect that experts will disagree with our layout, and hope that they will suggest how and why to make alterations. If such comments are reasonable, changes will be made. Additions are welcome too, especially if they come with detailed protocols! In the end, we intend this figure to be a living one, much like Wikipedia. Where available, protocols are underlined and linked below.

Buffers and Solutions

Tris-EDTA (TE)

Phosphate Buffered Saline (PBS)

Tris- Acetic Acid- EDTA (TAE)

Queen’s Buffer

Ricklefs’ Buffer

Casein Buffer

Bicarb/carb Buffer

Extraction Buffer

Cricket Hemolymph Anti-coagulant

 


 

Cell Counts and Cellular Activity

Total Haemocytes (invertebrate blood smears)

Total Leukocytes (vertebrate blood smears)

Lymphoid tissue mass

Sponge implantation

Bacterial and fungal killing assay using plates

Bacterial killing assay using a spectrophotometer

Phagocytosis assay

Encapsulation

Lipid analysis

In vitro Cell Proliferation Assay


Circulating Immune Molecules (antibodies, cytokines, etc.)

Antigen specific antibodies (indirect ELISA)

HL-HA assay for detection of natural antibodies

Haptoglobin

Complement

In vitro bacterial killing assay for insects

In vivo bacterial killing assay for insects

Zone of inhibition assay

Hemolymph coagulation assay

Nitric oxide assay

Real-time PCR for cytokines

Polymerase Chain Reaction (PCR) and DNA electrophoresis

C-reactive protein

Mannose-binding protein

Oxidative burst assay (kit required)

Anti-microbial peptides

Lysozyme-like activity assay (invertebrates)

Phenoloxidase (PO) and ProPO


Antigen Dosing and Challenging

General insect immune challenge techniques


Fever and Sickness Behavior

Telemetry (radio and internal)

Food consumption/anorexia

Fever


Swelling and Wound Repair

PHA swelling

Cutaneous wound healing


Antigen Detection

Sandwich ELISA

Blood endotoxin levels and clearance (kit required)

Antigen and parasite clearance

Plaque reduction neutralization test (PRNT)


Molecular techniques

Phenol-chloroform isoamyl alcohol (PCI) DNA extraction

RNA extraction

cDNA synthesis from RNA (invitrogen: SuperScript III First-Strand Synthesis System)

Cloning kit (Invitrogen: TOPO TA Cloning Kit)

PCR Clean-up (Qiagen: QIAquick PCR Purification Kit)

 

Please use these protocols freely, but we request that if a publication results from their use, the authors acknowledge this website (www.ecoimmunology.org), the author(s) of the protocol used, and the grant that supports this site (NSF-0947177) in the acknowledgements sections.  Also, please notify us at rdei.rcn@gmail.com of your publication so that we can list it on our Recent News page. 

Please contact us (rdei.rcn@gmail.com) if you have protocols you would like to share on this website.