Counting Hemocytes in Invertebrate Immune Assay

The number of circulating haemocytes was determined according to Urbański et al. [43 (link)]. Twenty-four hours after injection of EXT, the hemolymph sample (2 µL) was gently mixed with the 20 µL of the physiological saline solution and the anticoagulation buffer (4.5 mM citric acid and 9 mM sodium citrate; 5:1, v/v) and then placed on the Bürker chamber. The photos of 24 randomly selected squares were taken with a Zeiss Primostar microscope equipped with Axiocam 105 digital camera (Zeiss, Jena, Germany). To evaluate the number of haemocytes on the photos, cells were counted using ImageJ software (version 2., public domain, https://imagej.net/software/imagej2/ (accessed on 20 October 2022)). At least 13 individuals were used per treatment (Control—16, EXT 0.01%—14 and EXT 0.1%—13).