Dnp30 40 conjugated human serum albumin dnp hsa

Mice were sensitized in both ears with one intradermal injection per ear of 20 μl PBS containing 20 ng anti-dinitrophenyl (DNP) IgE (kindly provided by Dr. Fu-Tong Liu, then at University of California-Davis, Davis, Ca) (Liu et al., 1980 (link)) to sensitize mast cells, or received PBS only (using a 30 g needle on a one ml syringe; BD Biosciences) into both ears (Marichal et al., 2013 (link)). 18 hours later, the mice were anesthetized and treated with the ear skin and soft tissue (ESST) infection model. Immediately after ear pricking, 100 μl of 0.9% saline containing 100 μg DNP_30-40-conjugated human serum albumin (DNP-HSA; Sigma) was injected into the retro-orbital vein to induce mast cell degranulation in IgE-sensitized ears. Thickness of the non-infected ear was measured using a dial thickness gauge (model G-1A; Peacock Ozaki) in order to assess degranulation-mediated ear swelling. Ears and cervical lymph nodes were harvested after 20 hours as described above.

IgE-dependent PCA was induced in the ear pinna as described previously (2 (link)). Briefly, mice under isoflurane anesthesia were sensitized passively with IgE by intradermal injection of 20 ng of dinitrophenol (DNP)—specific IgE (α-DNP clone 26, using cells kindly provided by Fu-Tong Liu, University of California, Davis and D.H. Katz of LIDAK Pharmaceuticals), in 20 μL of HMEM-Pipes buffer (Sigma-Aldrich) in the left ear pinna; mice received 20 μL of vehicle intradermally in the right ear pinna as a control. The next day, mice were challenged through the retro-ortibal venus sinus with 100 μg of DNP_30-40—conjugated human serum albumin (DNP-HSA; Sigma-Aldrich) in 100 μL of saline.
Immediately before or at defined intervals after antigen challenge, ear thickness was measured with a dial thickness gauge (G-1A; Ozaki). Differences between left and right ear pinna were calculated (Δ ear thickness). Mice were sacrificed 6 hours after antigen challenge and ear pinnae were collected for histological analysis.