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This in vitro study aimed to find the best method of granulocyte isolation for subsequent labeling with multimodal nanoparticles (magnetic and fluorescent properties) to enable detection by optical and magnetic resonance imaging (MRI) techniques. The granulocytes were obtained from venous blood samples from 12 healthy volunteers. To achieve high purity and yield, four different methods of granulocyte isolation were evaluated. The isolated granulocytes were labeled with multimodal superparamagnetic iron oxide nanoparticles (M-SPIONs) coated with dextran, and the iron load was evaluated qualitatively and quantitatively by MRI, near-infrared fluorescence (NIRF) and inductively coupled plasma mass spectrometry (ICP-MS). The best method of granulocyte isolation was Percoll with Ficoll, which showed 95.92% purity and 94% viability. After labeling with M-SPIONs, the granulocytes showed 98.0% purity with a yield of 3.5 × 10 6 cells/mL and more than 98.6% viability. The iron-loading value in the labeled granulocytes, as obtained by MRI, was 6.40 ± 0.18 pg/cell. Similar values were found with the ICP-MS and NIRF imaging techniques. Therefore, our study shows that it is possible to isolate granulocytes with high purity and yield and labeling with M-SPIONs provides a high internalized iron load and low toxicity to cells. Therefore, these M-SPION-labeled granulocytes could be a promising candidate for future use in inflammation/infection detection by optical and MRI techniques.Hard-to-reach or occult inflammation and infection processes are clinically challenging. Their clinical diagnoses involve biochemical and radiological examinations, but these methods can produce false negatives [11][12][13][14][15]. To determine the localization, extent and severity of inflammation, imaging techniques such as computed tomography (CT) and magnetic resonance imaging (MRI) can be performed but have limitations in the early stages of the disease, as they are dependent on morphological changes [16][17][18]. Accurate and early diagnosis of inflammation and infection helps reduce mortality and morbidity and increase drug treatment success [19][20][21].Proinflammatory effects help leukocytes migrate from the blood vessel to the site of injury, due to increased vascular permeability through stimulation by cytokines and interleukins. There is an initial accumulation of granulocytes, mainly neutrophils, in the inflamed area and later of lymphocytes and macrophages [22][23][24]. Granulocytes are the most abundant subset of leukocytes [25] and are produced in the bone marrow, originating from myeloid precursor cells and undergoing a maturing process before they are able to perform their functions, mainly phagocytosis [25,26]. They differ from other cells in that they have granules with proteolytic enzymes that fight microorganisms or other inflammatory agents [25,27]. Granulocytes are considered frontline cells in inflammation and infection processes [28]. In many cases, to detect inflammation/infection, it is necessary to label isolated granulocyte...