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ANTIHYPERLIPIDEMIA AGENTS Plasma lipids Transported in bloodstream in form of macromolecular complexes of lipid and known as lipoproteins Two major clinical importance/sequelae of high lipid Acute pancreatitis atherosclerosis Hyperlipoproteinemia Hyperlipidemia Lipoprotein disorders Primary hypertriglyceridemias Primary chylomicronemia Familial hypertriglyceridemia Familial combined hyperlipoproteinemia Familial dysbetalipoproteinemia Primary hypercholesterolemias Famimial hypercholesterolemia Familial ligan-defective apolipoprotein B Familial combine hyperlipoproteinemia Lp(a) hyperlipoproteinemia Secondary hyperlipoproteinemia Lipid-lowering drugs Several drugs are used To decrease plasma LDL-cholesterol Drug therapy is only one approach Dietary measures are the first [...] | 19th January, 2010 | More News
Isolation of neutrophils
Materials
Citrate (0.32%) as anticoagulant
4.5% dextran T-500 in PBS (sterile), room-temperature
Ficoll-Conray solution (density, 1.077 g/ml), sterile
PBS
20 x concentrated PBS, ice-cold
Turk’s solution
15-ml conical plastic centrifuge tubes
Refrigerated low-speed centrifuge
Lucigenin stock: 10 mM – final concentration of 0.1 mM.
Buffer Saline Albumin
Stimulant stock: 1mg PMA /ml
CL Monitor: Berthold Multi-Biolumat LB 9505 C Luminometer
Measuring vials
Neutrophil source: were blood obtained as above
Procedure
White blood cell separation from erythrocytes by dextran sedimentation
Five ml of blood was drawn from a healthy volunteer into a syringe containing preservative citrate. Mixed blood with 2.5 ml of dextran solution in a plastic cylinder or 15-ml centrifuge tube and incubated in upright position about 10 minutes at room temperature. Leukocyte-rich plasma (upper layer) was aspirated and saved. Then, PBS was added up until 12 ml and centrifuged for 10 minutes at 1200 rpm, 25 Celsius. The supernatant was discarded and 500µl of PBS was added to the pallet.
Removal of residual red blood cell (RBC) by hypotonic lysis
RBCs were removed by subjecting cells into hypotonic solution. Leukocyte /RBC suspension was re-suspended with 9.5 ml of water for exactly 25 seconds. At the end of this period, isotonicity was restore by adding 20 x concentrated PBS and centrifuged for 10 minutes at 900 rpm, 25 Celsius. The supernatant was discarded and 1 ml of PBS was added on to the pallet.
Removal of mononuclear cells by Ficoll-Conray solution centrifugation and count
Cell suspension was layered over 3 ml Ficoll-Conray solution using a pipet or a syringe fitted with a long blunt-end needle. Care was taken to preserve a sharp interface between the Ficoll-Conray solution and the cell suspension. The mixture was centrifuged for 20 minutes at 2000 rpm, 20 Celsius. The middle (lymphocyte/monocyte) layer was aspirated as well as the Ficoll-Conray layer, leaving pellet, that is the neutrophils. The neutrophils were suspended in 2 ml PBS. The neutrophil suspension was then washed once or twice until the cell pellets look white by 3-minutes centrifugation at 1200 rpm, 5 celsius. The supernatant was discarded, and the pellets were re-suspended in 1 ml ice-cold PBS. The cells count was determined on a microscope by using a glass counter chamber.
About the Author
Specialized in Tropical Medicine and Research on men health especially in osteoporosis