Nuscript Author Manuscript Author Manuscript2.3.four.7.1.three.two Flow α adrenergic receptor Antagonist Molecular Weight cytometric detection of cell death in human granulocytes: Human granulocytes can easily be obtained via density gradient centrifugation of human blood. Numerous distinctive protocols have already been published, with some involving dextran sedimentation of RBCs. The protocol we describe here omits the lengthy dextran sedimentation step with out affecting the purity of the granulocyte fraction. 1. A total of 20 mL of anti-coagulated blood is diluted with 15 mL PBS and gently layered on top rated of 15 mL Lymphoflot. Cells are separated via centrifugation at 300 g for 30 min without having break. The granulocytes layer straight on top of the RBCs (whitish veil) and are collected and washed after in PBS. Note that this fraction consists of primarily neutrophils and eosinophils, whereas basophils sediment within the PBMC fraction. The cell pellet is resuspended in 200 L of PBS. Hypotonic lysis of erythrocytes is performed by addition of 36 mL of icecold water for 20 s. Physiological osmolality is re-obtained by addition of four mL of 10PBS. The granulocytes are resuspended in RPMI-1640 supplemented with one hundred U/mL penicillin/streptomycin, 2 mM glutamine, and 10 heat-inactivated FCS and 25 mM HEPES at a concentration of 2 106 cells/mL and cultivated at 37 /5 CO2. As a consequence of the short life span of granulocytes, detectable cell death will occur in much less than 12 h. Cell death is assessed by harvesting of cells through centrifugation at 300 g for five min and resuspension at a concentration of 1 106 cells/mL in HBSS2.3.four.Eur J Immunol. Author manuscript; accessible in PMC 2020 July 10.Cossarizza et al.Pagesupplemented with two heat inactivated FCS, 100 ng/mL PI, and 1 g/mL ANXV. Staining is performed on ice for 30 min. 5. Without the need of an further washing step, samples are straight subjected to FCM evaluation. Note that washing will not be advised as this could result in the loss of subcellular particles and compromise integrity of apoptotic cells. Flow cytometric detection of particle uptake in human granulocytes A total of 20 mL of anti-coagulated blood is diluted with 15 mL PBS and gently layered on top of 15 mL Lymphoflot. Cells are separated through centrifugation at 300 g for 30 min without break. The granulocytes layer directly on prime from the RBCs (whitish veil) and are collected and washed when in PBS. Note that this fraction contains primarily neutrophils and eosinophils, whereas basophils sediment inside the PBMC fraction. The cell pellet is re-suspended in 200 L of PBS. Hypotonic lysis of erythrocytes is performed by addition of 36 mL of ice-cold water for 20 s. Physiological osmolality is re-obtained by addition of 4 mL of 10PBS. The granulocytes are re-suspended in in HBSS supplemented with two heat inactivated FCS. A total of 20 g/mL micro monosodium urate crystals and 250 g/mL Lucifer Yellow are added and cells are incubated at 37 /5 CO2 for a variety of time points. Cells are collected and without the need of more washing directly subjected to FCM evaluation. MaterialsAuthor Manuscript Author Manuscript Author Manuscript Author Manuscript7.1.3.three 1.two.188.8.131.52.7.1.four.1 Reagents: HBSS, calcium, magnesium, no phenol red (ThermoFisher Met Inhibitor medchemexpress Scientific, 14025050) Lymphoflot (Bio-Rad, #824012) Lucifer Yellow CH (ThermoFisher Scientific, L453) RPMI 1640 Medium (ThermoFisher Scientific, 21875034) L-Glutamine (ThermoFisher Scientific, 25030081) Penicillin treptomycin (ThermoFisher Scientific, 15140122) HEPES (ThermoFisher Scientific, 15630056) Fetal Calf Serum (Biochrom,.