Nuscript Author Manuscript Author Manuscript2.three.four.7.1.three.2 Flow cytometric detection of cell death in human granulocytes: Human granulocytes can easily be obtained via density gradient centrifugation of human blood. Several 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 having 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 top of 15 mL Lymphoflot. Cells are separated by way of centrifugation at 300 g for 30 min without the need of break. The granulocytes layer directly on best from the RBCs (whitish veil) and are collected and washed after in PBS. Note that this fraction contains mostly neutrophils and eosinophils, whereas basophils sediment inside the PBMC fraction. The cell pellet is SGLT2 Inhibitor manufacturer 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, two mM glutamine, and 10 heat-inactivated FCS and 25 mM HEPES at a concentration of two 106 cells/mL and cultivated at 37 /5 CO2. Due to the brief life span of granulocytes, detectable cell death will take place in much less than 12 h. Cell death is assessed by harvesting of cells by means of centrifugation at 300 g for 5 min and PKCĪ¶ Inhibitor Compound resuspension at a concentration of 1 106 cells/mL in HBSS2.3.4.Eur J Immunol. Author manuscript; available in PMC 2020 July ten.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. five. Without having an added washing step, samples are straight subjected to FCM analysis. Note that washing just isn’t 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 major of 15 mL Lymphoflot. Cells are separated through centrifugation at 300 g for 30 min with no break. The granulocytes layer directly on best with the RBCs (whitish veil) and are collected and washed after in PBS. Note that this fraction includes mainly neutrophils and eosinophils, whereas basophils sediment in 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 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 numerous time points. Cells are collected and without the need of additional washing directly subjected to FCM evaluation. MaterialsAuthor Manuscript Author Manuscript Author Manuscript Author Manuscript7.1.3.3 1.2.3.4.7.1.7.1.4.1 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 (Biochrom,.
