May gr nwald giemsa

Human whole blood from healthy volunteers was collected into ethylenediaminetetraacetic acid (EDTA)-containing tube. Neutrophils were isolated by density gradient centrifugation using Polymorphprep (Alere Technologies, Germany) according to the manufacturer’s instructions. Neutrophils were re-suspended with Roswell Park Memorial Institute (RPMI) 1640 (Gibco/Life Technologies, MA, USA) without phenol red supplemented with 1% fetal bovine serum (FBS; Gibco/Life Technologies, MA, USA) for normal culture conditions. Alternatively, neutrophils were cultured under serum-free (i.e., RPMI-1640 with 0% FBS) or albumin-depleted (i.e., RPMI-1640 with 1% FBS without albumin—see “Albumin depletion from serum or plasma”) conditions. For the replenishment of albumin, bovine serum albumin (BSA; BioShop Canada Inc., Canada) dissolved in phosphate buffered saline (PBS) was freshly prepared and added to albumin-free media at a final concentration of 0.02 g per dL, matching the albumin concentration in the condition of 1% FBS. Neutrophil purity was established to be routinely >90%, as assessed by May-Grünwald Giemsa (Sigma-Aldrich, Canada) staining.

Total leukocyte number was calculated in each SF sample staining with Türk’s solution (Sigma-Aldrich, St Louis, Missouri, USA) and counting by LUNA-II™ Automated Cell Counter (Logos Biosystems, Gyeonggi-do, South Korea). Haemorrhagic SF samples were discarded (1 from MSU group, and 2 from MSU+MSC group). Leukocyte differential count was performed in the SF from each knee joint cavity. SF smears were fixed in methanol and subsequently stained with May-Grünwald Giemsa (Sigma-Aldrich, St Louis, Missouri, USA) staining. Ten different pictures in each sample were obtained with a Leica DM 6000 LED instrument (Leica, Microsystems, Inc. Buffalo Grove, IL, USA) in order to calculate the percentage of polymorphonuclear cells (PMNC) and mononuclear cells (MNC) present in each sample.

The peripheral blood samples of each animal were used to measure the total leukocyte count by Neubauer chamber after 1:20 dilution in Turkey’s solution. Diluted blood samples were transferred to the hemocytometer chamber. Calculations were made manually in 25 hemocytometer squares, using a 40× objective lens. Thereafter, blood samples were smeared on a glass slide and stained with May-Grünwald-Giemsa (Sigma-Aldrich Chemie GmBh, Steinheim, Germany) in order to perform differential leucocyte count. Neutrophils, lymphocytes, and monocytes were counted using a light microscope (Nicon, Tokyo, Japan) under a 100× objective lens. Then 300 cells were counted and used for percentage calculations.

Mice were euthanized by CO₂ overdose using Prodigy Lab Control Unit (Smartbox) and bronchoalveolar lavage fluid (BALF) was performed as previously described (7 (link)). Differential cell counts were routinely performed on cytospin preparation (Cytospin 3, Thermo Shandon) after May-Grünwald Giemsa staining (Sigma-Aldrich, St Louis, MO) according to the manufacturer’s instructions and at least 200 cells were counted using standard morphological criteria.

Cytologic samples from each case were stained with May–Grünwald–Giemsa (Sigma-Aldrich, Merck, Darmstadt, Germany). MCTs were classified according to Kiupel’s two-tier grading system proposed by Camus et al. [7 (link)] for cytologic specimens. In this system, samples exhibiting poor granulation and/or meeting at least two malignancy criteria (such as the presence of mitotic figures, nuclear pleomorphism, binucleation or multinucleation, or marked anisokaryosis) were classified as high grade. Conversely, well-granulated samples without the mentioned malignancy features were categorized as low grade.

Cells were resuspended in culture media and concentrated by cytospin centrifugation at 700g for 5 min onto SuperFrostPlus slides (ThermoFisher Scientific) using a Shandon Cytospin 3 cytocentrifuge. Slides were fixed for 3 min in cold methanol and stained with May-Grünwald-Giemsa (Sigma). Images were captured using a Leica DM5000b microscope in conjunction with a × 63 oil-immersion lens and an Olympus DP72 camera.