Cabr2

Samples of 200 μL of peritoneal fluid were taken from mice injected with 5 mL of RPMI at 37 °C after a 1.5 min massage of the abdomen. Samples were evaluated after being cooled to 4 °C, before being frozen at −20 °C until use. Attenuated Total Reflectance (ATR) analyses of the initial chitosan and peritoneal samples were recorded on a Genesis II FTIR spectrophotometer (Mattson, Madison, WI, USA) equipped with an ATR device (MIRacleTM PIKE Technologies, Madison, WI, USA) and a zinc selenide (ZnSe) crystal. For FTIR analysis, the initial chitosan and peritoneal samples were poured on the spectrophotometric windows from CaBr₂ (Sigma-Aldrich) and evaporated at room temperature (25 °C) for 24 h. FTIR spectra were collected in the wavenumber range between 4000 cm⁻¹ and 600 cm⁻¹, at a resolution of 4 cm⁻¹ using 64-times scanning [69 (link)].

Samples of 200 µL of brain homogenate were diluted with 200 µL PBS with protein inhibitors. Samples were cooled to 4 °C, before being frozen at −20 °C until use. Attenuated Total Reflectance (ATR) analyses of the initial chitin were recorded on a Nicolet iS10 spectrophotometer (Thermo Scientific, Waltham, MA, USA) equipped with an attenuated total reflectance mode with a diamond as the crystal. The spectra were processed using Omnic 9.3.30 (Thermo Scientific, Waltham, MA, USA). For FTIR analysis, the initial chitin and N-acetyl-glucosamine and brain samples were placed on the spectrophotometric windows made of CaBr₂ (Sigma-Aldrich, Poznań, Poland) and allowed to evaporate at room temperature (25 °C) for 24 h. FTIR spectra were collected in the wavelength range between 4000 cm⁻¹ and 500 cm⁻¹, at a resolution of 2 cm⁻¹ using 100-times scanning.
All measurements were made on a CaF₂ plate, onto which a vortexed sample drop was applied. After drying, the measurement was performed at room temperature, collecting spectra at several different locations in the sample. The results were analysed in relation to the two standards used, namely, N-acetyl-glucosamine by Sigma Aldrich (Poznań, Poland) and chitin by PRIMEX (Siglufjordur, Iceland). Finally, when compared with spectra from the literature [100 (link),101 (link),102 (link),103 (link)], there is a great similarity to these spectra.