Neutrophils (2.5 × 106 ml− 1), either in the presence or in the absence of erythrocytes and/or adenosine (10− 8 M or 10− 5 M), were resuspended in RPMI with 10% foetal bovine serum with and without ticagrelor (10− 5 M) in a final volume of 100 μl in a 96-well plate. Assays were performed in duplicate. To allow visualisation of neutrophil phagocytosis by microscopy, it was necessary to dilute the erythrocytes, which were therefore resuspended at 12.5 × 106 ml− 1. Heat-killed, opsonized Streptococcus pneumoniae was added to achieve a multiplicity of infection (MOI) of 20 and incubated for 30 min (37 °C, 5% CO2). Cytocentrifuge slides were prepared from the cell suspension using a Cytospin machine (Shandon, Thermo Scientific, Waltham, MA) and stained with modified Giemsa based stains (Differentiation-Quik, Reagena, Toivala, Findland). The percentage of neutrophils containing phagocytosed S. pneumoniae was determined by assessment of 300 neutrophils by light microscopy. Neutrophil phagocytic index was then determined using the following formula: (total number of engulfed bacteria / total number of counted neutrophils) × (number of neutrophils containing engulfed bacteria / total number of counted neutrophils) [20] (link).
Cytospin machine
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Cytospin machine is a laboratory instrument used for the preparation of cell samples for microscopic examination. Its core function is to concentrate cells onto a microscope slide, allowing for a more detailed analysis of the cellular morphology and composition.
Automatically generated - may contain errors
Lab products found in correlation
Rbc lysis buffer, by BD (1 mentions)
Hemacolor staining kit, by Merck Group (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Hema 3 stain kit, by Thermo Fisher Scientific (1 mentions)
Canto 2, by BD (1 mentions)
Model bx51, by Olympus (1 mentions)
Propidium iodine pi, by Merck Group (1 mentions)
5 protocols using cytospin machine
1
Neutrophil Phagocytosis of Streptococcus pneumoniae
2
Neutrophil Quantification in Murine Bronchoalveolar Lavage
3
Lung Inflammation Assessment Protocol
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To detect the inflammation occurred in the lung, we counted the number of inflammatory cells in BALF by cytospin. In short, the cell suspension was centrifuged for 5 minutes at a 300 rpm in a Cytospin machine (Thermo). The numbers of macrophages and neutrophils were counted by a hemocytometer. The inflammasome activity in the lung was detected by using a Caspase 1 Inflammasome Assay Kit (Thermo).
4
Quantification of Mouse Lung Inflammatory Cells
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The main trachea of a mouse was cannulated, followed by a wash of the lung with 1 mL of PBS two times to collect bronchoalveolar lavage (BAL) fluids. Cells in the BAL fluids were counted and prepared for smears using a Cytospin machine (Thermo, Waltham, MA, USA). The slides were air-dried and then stained with Hema-3 Stain kit (Fisher Scientific, Hampton, NH, USA), which contained a cell fixative and eosin Y stain. The number of eosinophils, monocytes/macrophages (mon/Mφ), and lymphocytes per 200 cells were counted and differentiated based on cellular morphology and staining characteristics. Cells in the BAL were also analyzed by flow cytometry (Canto II, BD) for eosinophils with fluorescence-conjugated mAbs.
5
Shrimp Hemolymph Necrosis Assay
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Hemolymph (100 µl) was withdrawn from the ventral sinus of each shrimp and diluted with 100 µl of anticoagulant solution (30 mM trisodium citrate, 340 mM sodium chloride, and 10 mM EDTA, at pH 7.5 with the osmolality adjusted to 718 mOsm/kg with 115 mM glucose). LPS, βG and PG solutions were prepared with shrimp salt solution (SSS, anticoagulant solution without EDTA) to make final concentrations of 0.1, 0.5, and 1.0 mg/ml. Details of hemolymph sampling, treatment with LPS, βG and PG, and cell necrosis were conducted following previously described procedures [13] (link). Briefly, 100 µl of each test solution (LPS, βG, PG) was added to hemolymph sample and incubated for 30 min. A group that 100 µl of SSS was added to hemolymph sample and incubated for 30 min served as controls. The solution was added to 10 µl RNase (1 mg/ml) and incubated for 30 min at 37°C, and 20 µl of propidium iodine (PI, Sigma) solution then added and samples incubated for 15 min at room temperature. Fifty microliters of the mixed solution were spread on a slide glass and centrifuged with cytospin machine (Thermo, Shandon, UK) at 1000 rpm for 3 min. Permanent slides were made and examined under a fluorescent microscope (Model BX 51, Olympus, Tokyo, Japan) for cell death (necrosis) [22] . Hemocytes were also examined under a fluorescent microscope to determine the percentage of necrotic cells.
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