Nuscript Author Manuscript Author Manuscript2.3.4.7.1.3.2 Flow cytometric detection of cell death in human granulocytes: Human

Nuscript Author Manuscript Author Manuscript2.3.4.7.1.3.2 Flow cytometric detection of cell death in human granulocytes: Human granulocytes can very easily be obtained by way of density gradient centrifugation of human blood. Various various protocols happen to be published, with some involving dextran sedimentation of RBCs. The protocol we describe right here omits the lengthy dextran sedimentation step devoid of affecting the purity of your granulocyte fraction. 1. A total of 20 mL of anti-coagulated blood is diluted with 15 mL PBS and gently layered on major of 15 mL Lymphoflot. Cells are separated by way of centrifugation at 300 g for 30 min devoid of break. The granulocytes layer straight on major of 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 in 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, two mM glutamine, and ten heat-inactivated FCS and 25 mM HEPES at a concentration of 2 106 cells/mL and cultivated at 37 /5 CO2. As a result of the brief life span of granulocytes, detectable cell death will happen in much less than 12 h. Cell death is assessed by harvesting of cells via centrifugation at 300 g for 5 min and resuspension at a concentration of 1 106 cells/mL in HBSS2.3.four.Eur J FGF-8 Proteins Formulation Immunol. Author manuscript; available in PMC 2020 July 10.Cossarizza et al.Pagesupplemented with 2 heat inactivated FCS, 100 ng/mL PI, and 1 g/mL ANXV. Staining is performed on ice for 30 min. 5. With out an added washing step, samples are directly subjected to FCM evaluation. Note that washing is just not suggested as this can lead to 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 leading of 15 mL Lymphoflot. Cells are separated by way of centrifugation at 300 g for 30 min with no break. The granulocytes layer straight on leading in the RBCs (whitish veil) and are collected and washed once in PBS. Note that this fraction includes 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 four mL of 10PBS. The granulocytes are re-suspended in in HBSS supplemented with 2 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 various time points. Cells are collected and without having additional washing directly subjected to FCM evaluation. MaterialsAuthor Manuscript Author Manuscript Author Manuscript Author Manuscript7.1.3.3 1.2.three.four.7.1.7.1.4.1 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 (FGF-10 Proteins custom synthesis Biochrom,.