NPY Y4 receptor Agonist Gene ID Nuscript Author Manuscript Author Manuscript2.three.4.7.1.3.two Flow cytometric detection of cell death in human granulocytes: Human granulocytes can conveniently be obtained by way of density gradient centrifugation of human blood. Many distinct protocols have already been published, with some involving dextran sedimentation of RBCs. The protocol we describe right here omits the lengthy dextran sedimentation step without affecting the purity in the granulocyte fraction. 1. A total of 20 mL of anti-coagulated blood is diluted with 15 mL PBS and gently layered on best of 15 mL Lymphoflot. Cells are separated through centrifugation at 300 g for 30 min without the need of break. The granulocytes layer straight on leading on the RBCs (whitish veil) and are collected and washed after in PBS. Note that this fraction consists of mostly 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 4 mL of 10PBS. The granulocytes are resuspended in RPMI-1640 supplemented with 100 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. Because of the quick life span of granulocytes, detectable cell death will take place in significantly 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.three.4.Eur J Immunol. Author manuscript; accessible in PMC 2020 July 10.Cossarizza et al.Pagesupplemented with 2 heat inactivated FCS, one hundred ng/mL PI, and 1 g/mL ANXV. Staining is performed on ice for 30 min. five. Devoid of an further washing step, samples are directly subjected to FCM analysis. Note that washing just isn’t recommended as this can 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 prime of 15 mL Lymphoflot. Cells are separated via centrifugation at 300 g for 30 min with out break. The granulocytes layer directly on top rated from the RBCs (whitish veil) and are collected and washed when in PBS. Note that this fraction includes mainly 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 RSK3 Inhibitor Compound Yellow are added and cells are incubated at 37 /5 CO2 for various time points. Cells are collected and with no additional washing directly subjected to FCM analysis. MaterialsAuthor Manuscript Author Manuscript Author Manuscript Author Manuscript7.1.3.3 1.two.3.four.7.1.7.1.four.1 Reagents: HBSS, calcium, magnesium, no phenol red (ThermoFisher 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,.