Z4250

Zymosan (20μl 5mg/mL Sigma: Z4250) was injected into the hindpaw and GCaMP fiber photometry was performed as above during von Frey and Hargreave’s assays at baseline and the peak of sensitivity (4 hours post injection).

PDFGRb-P2A-CreERT2 × ROSA26 td-Tomato transgenic mice were used for the zymosan-induced arthritis experiment. To induce Cre activity, adult transgenic mice (14-week-old) were first orally treated with tamoxifen at a dose of 50 mg/kg of body weight for three consecutive days. Two days after the last dose of tamoxifen, to induce arthritis, mice received an intra-articular injection of zymosan (180 μg) (Sigma-Aldrich, Z4250) into one knee joint, and the contralateral knee was injected with the same volume of saline as control. Joints with both femur and tibia were collected for analysis after 48 hours after injection.

Imidacloprid (37894-100 mg, Sigma-Aldrich™) and amitraz (45323, Sigma-Aldrich™) stock solutions were dissolved in WH2 medium, each to two concentrations, 40 and 200 µg/ml, while zymosan A (Z4250, Sigma-Aldrich™) stock solution had a concentration of 4 µg/ml. All stock solutions were sonicated for 30 min in a water-bath sonicator before usage to ensure dissolution of the pesticides and zymosan A. Treatments and/or WH2 medium were added to 24-well tissue culture plates (92024, TPP™) containing 100 µl of diluted hemolymph to reach a total volume of 400 µl. In addition to the treatment control, the final concentrations of treatments were 10 or 50 µg/ml for either Imidacloprid or amitraz single exposures. Pesticide mixtures were set to the final concentrations of 10 + 10 µg/ml, 10 + 50 µg/ml, or 50 + 10 µg/ml of Imidacloprid and amitraz. All pesticide treatments were done in either no zymosan or with 1 µg/ml zymosan. Plates were sealed with sterilized sealing tape and incubated at 20°C in the dark for 18 h. All conditions were made in triplicates (n = 3).

The mouse model of peritonitis was used to image endogenous H₂O₂. Briefly, the C57BL/6J mice were injected intraperitoneally with 200 μl ZA (2 mg ml⁻¹, Z4250, Sigma-Aldrich) for 48 h. For the inhibitor study, mice were treated 200 μl GSH (200 mg kg⁻¹) for 24 h by intraperitoneally after ZA treatment. The control mice were treated only using 200 μl saline. After 48 h of the ZA treatment, anesthetized mice (2% isoflurane in oxygen) were treated with intraperitoneal injection of 200 μl CDGA (200 μg ml⁻¹ based on CDs). Then, the mice were immediately put into the IVIS imaging chamber (Xenogen, Alameda, CA) to acquire luminescent signals with an exposure time of 3 min at every 5 min with open filter at 675 nm. The phosphate buffer saline (PBS) was used in control group.

This assay measures tested the capacity of phagocytes (mainly heterophils) in toad whole blood to engulf zymosan particles. We added 40 µl of whole blood to 760 µl of sterile amphibian Ringers to produce a 1 : 20 dilution. We then added 240 µl of the diluted blood to duplicate wells of a 96-well microassay plate along with 30 µl of luminol (A8511, Sigma) solution and 10 µl of zymosan (Z4250, Sigma) solution. A control well for each sample contained 240 µl of diluted blood, and 30 µl luminol but with 10 µl of PBS instead of zymosan. Immediately upon addition of zymosan to the wells, the plate was placed in a luminometer (FluoroStar Optima; BMG Labtech, Ortenberg, Germany) and luminescence was read at 5 min intervals over 180 min. Higher luminescence values indicate higher rates of phagocytosis occurring in the sample.