Adipocytokine test v1 26/Sep/2016 RIKENMPP_020b_002 test 2 1 testSt test modification

Levels of serum adipocytokines are an important indicator of lifestyle diseases, including diabetes and arteriosclerosis.

These procedures are applicable to the mouse phenotyping platform at the RIKEN BRC. The tests should be performed by trained staff members who understand the safety and hygiene rules required by RIKEN. Any queries, comments, or suggestions, relating either to this SOP in general or to a specific problem encountered during a procedure, should be addressed to the Blood Tests Unit Leader. Any deviation from this protocol must be reported to the Blood Tests Unit Leader.

Testing is carried out in accordance with common animal handling procedures. Coveralls (in the rearing facility) or laboratory coats (in the laboratory facility) must be worn with gloves at all times in the work area. If animals are dropped on the floor, treat them in accordance with the safety and hygiene guidelines of the rearing facility at RIKEN. If something unexpected happens during the primary screening, it must be quickly reported to the Blood Tests Unit Leader and recorded.

Follow the manual for the multiplex suspension array system (Bio-plex, Bio-Rad Laboratories) for quality control.

Bio-plex (Bio-Rad Laboratories) Sonicator Shaker Magnetic washer

Adipocytokine assay kits Mouse Diabetes 8-plex Panel (171AF7001M) for Ghrelin, GIP, GLP-1, Glucagon, Insulin, Leptin, PAI-1, Resistin Mouse Adiponectin kit (171F002M) for Adiponectin Bio-plex reagents (see the manufacturer's manual).

Prepare the sera according to the SOP for Blood Collection for Adipocytokine Analysis. Follow the manuals from the adipocytokine assay kits to assay the samples. It is possible to use half the amount of sample and reagents described in the protocols. Follow the manual for the Bio-plex system to make the measurements.

Parameters 1. Adiponectin 2. Ghrelin 3. GIP 4. GLP-1 5. Glucagon 6. Insulin 7. Leptin 8. PAI-1 9. Resistin

Measurement parameters were changed. Glucagon was removed and measurement kit was changed (Ver.1.01). Before : Insulin, Amylin, Leptin, Glucagon, GLP-1, MCP-1, IL-6, TNF-a, tPAI-1, Resistin, Adiponectin (11 parameters) After : Insulin, Amylin, Leptin, GLP-1, MCP-1, IL-6, TNF-a, tPAI-1, Resistin, Adiponectin (10 parameters) Before : Mouse Endocrine Panel (Millipore, MENDO-75K) After : Mouse Metabolic Disease Panel (Millipore, MMHMAG-44K) Measurement parameters were changed. Ghrelin and GIP and Glucagon were added. TNF-α and MCP-1 and IL-6 and Amylin were removed. Measurement kits were changed (Ver.2.00). Before : Insulin, Amylin, Leptin, GLP-1, MCP-1, IL-6, TNF-a, tPAI-1, Resistin, Adiponectin (10 parameters) After : Insulin, Leptin, GLP-1, PAI-1, Resistin, Adiponectin, Ghrelin, GIP, Glucagon (9 parameters) Before : Mouse Metabolic Disease Panel (Millipore, MMHMAG-44K), Mouse Adipokine Magnetic Bead Panel (MADKMAG-71K) After : Mouse Diabetes 8-plex Panel (171AF7001M) Before : Mouse Adiponectin Magnetic Bead (MADPNMAG-70K-01J) After : Mouse Adiponectin kit (171F002M)