were maintained in dechlorinated tap water at 25?C. clinical research and is a routine technique for analysis in humans, fisheries, and domestic animals. Recently, haematological parameters have been recognized as a useful assessment tool for analysing sub-mammalian exotic animals, such as amphibians1. The study of the African clawed frog, has some disadvantages as an experimental model. For example, it is an allotetraploid species, and becomes sexually mature 10C24 months post metamorphosis5. In contrast, the western clawed frog has a smaller diploid genome and a shorter generation time, making it advantageous to study the over genome has recently been fully sequenced6. However, the previously published haematological parameters of cannot account for the differences between strains of and as animal models, since they possess nucleated blood cell types including erythrocytes, leukocytes, and thrombocytes, as well as their unclassified progenitors2,11,12. Additionally, using the T12 monoclonal antibody targeting thrombocytes13, we were able to distinguish thrombocytes from other types of blood cells. Fluorescence analysis can be performed qualitatively using fluorescence microscopy or quantitatively using flow cytometry (FCM). Fluorochromes can bind to DNA and RNA separately, thereby enabling independent labelling. For example, murine hematopoietic stem Mepixanox cells (HSCs) were stained using Hoechst 33342, a DNA-bound fluorochrome that emits two wavelengths (Hoechst Blue and Hoechst Red), to distinguish side-population (SP) cells14. Cell classification by supravital cell staining with acridine orange (AO) was first reported in the 1960s15,16. Classically, this metachromatic fluorochrome has been used to rapidly stain DNA and RNA independently, based on AO emissions of green fluorescence upon binding to double-stranded DNA, and red fluorescence upon binding to single-stranded Mepixanox RNA. A previous study evaluated lymphocytes from human peripheral blood (PB) by fluorescent microscopy using AO-stained preparations17. Nowadays, it is known that AO accumulates within lysosomes and azurophilic granules of living cells and emits red fluorescence18C20. This type of staining enables clinicians to diagnose patients E1AF with chronic lymphocytic leukaemia, pertussis, hypogammaglobulinemia, acute leukaemia, uraemia, and other malignancies, as well as to distinguish Mepixanox human reticulocytes from erythrocytes. Moreover, morphological abnormalities present in human erythrocytes, such as red blood cell fragments and large platelets can be detected via FCM with AO staining21. In this study, six parameters were used to identify blood cell types: forward-scattered light (FSC), side-scattered light (SSC), nucleic acid and intracellular granule information obtained from green (F530) and red (F695) fluorescence intensity, cellular red fluorescence intensity (F695/FSC), and cellular green fluorescence intensity (F530/FSC). This analysis method has the potential to classify and separate blood cells, and to identify haematological abnormalities in sub-mammalian species where automated blood counting is as yet not possible. Our study Mepixanox will also enable researchers to characterize the haematological features of various animals, including genetically modified frogs and fish. Results The molar extinction coefficient of haemoglobin in haemoglobin was purified, and SDS-PAGE was performed. Monomeric (14?kDa) and dimeric (30?kDa) Hbs were detected. M: Molecular marker; N: Non-reduction condition; R: Reduction condition. (B) The absorbance at 535?nm of SLS-Hb was measured and the molar extinction coefficient was calculated. Bar indicates the mean of the extinction coefficient (n?=?66). (C,D) The fluorescence of purified haemoglobin was measured. (C) Excitation 485/20?nm, emission 528/20?nm. (D) Excitation 485/20?nm, emission 590/35?nm. Optimal time course and AO concentration for staining blood cells The relative fluorescence intensity (RFI; see Methods) rapidly increased during the first 10?min, then gradually increased until.