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Lymphocyte separation medium

Manufactured by Thermo Fisher Scientific
Sourced in Canada

Lymphocyte separation medium is a sterile, ready-to-use liquid medium used to isolate and purify lymphocytes from whole blood or other biological samples. It utilizes a density gradient separation technique to selectively separate lymphocytes from other blood cells.

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12 protocols using lymphocyte separation medium

1

Isolation and Analysis of Leukocytes

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Leukocytes were isolated from serially collected peripheral blood samples, spleens or liver grafts. Splenocytes were isolated using standard protocol. To obtain single cell-suspensions from cardiac or liver grafts, tissue was cut into small pieces (2 mm) and digested with collagenase IV (Worthington Biochemical Corporation, Lakewood, NJ). The resulting suspension was run through a 70 μm filter and washed with PBS. After centrifugation, the leukocytes were purified using lymphocyte separation medium (Fisher Scientific, Pittsburgh, PA). Phenotypical analysis was performed by using several panels of fluorescein-labeled anti-mouse mABs (against CD3, CD4, CD8, Ly6G, Ly6C, MHCII, CCR2, CXCR1, CD80, CD86) or rat monoclonal antibodies (CD3, CD4, or CD8). All antibodies were purchased from BD Biosciences. Frequencies of T-regulatory cells were identified using APC conjugated CD4, FITC conjugated-CD25, and PE conjugated-Foxp3 (Biolegend) monoclonal antibodies according to the manufacture instructions. Staining was performed with antibodies above (1 μg/106 cells) at 4 °C for 30 min, washed, and analyzed by FACS (BD Biosciences).
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2

Isolation and Differentiation of Human PBMCs

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Human peripheral blood mononuclear cells (PBMC) were isolated by lymphocyte separation medium (Fisher Scientific, Waltham, MA) density centrifugation from whole blood donated by healthy volunteers (Carter Blood Care, Fort Worth, TX). Monocytes were enriched from freshly isolated PBMC using magnetic-activated cell sorting CD14+ beads and LS Columns (Miltenyi Biotec, Auburn, CA), yielding an average 98% purity. To differentiate PBMC into MDM, PBMC were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Carlsbad, CA) with 10% heat-inactivated pooled human serum (Atlas Biologicals, Fort Collins, CO), 50 μg/ml gentamicin (Life Technologies), 10 μg/ml ciprofloxacin (Sigma, St. Louis, MO) and 50 ng/ml macrophage-colony stimulating factor for two weeks. MDM were cultured as adherent cells in 48-well plates (4×105/well) or 6-well plates (3×106) for virus infection or as non-adherent cultures in teflon flasks (2×106 cells/ml, 150×106 cells in flask) for transfection assays. Cultures were maintained by half-media exchange twice weekly.
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3

Isolation of Immune Cell Populations

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Spleen, lymph nodes, thymus and Peyer’s patches were excised from mice, and single cell suspensions were obtained by teasing the organs through a nylon mesh. Isolation of single cells from the kidneys was modified from a previously described procedure [7 (link)]. In brief, perfusion of the kidneys was performed with 30 ml of pre-warmed phosphate buffered saline (PBS). Then kidneys were digested with collagenase type 4 (100 µg/ml) (Worthington Biochemical Corp., Freehold, NJ) in Hank’s Balanced Salt Solution (HBSS) for 30 min (37 °C). Single cells of kidneys were isolated the same way as splenocytes and cell suspensions were subjected to density separation to eliminate the epithelial or tubular cells of the kidneys using lymphocyte separation medium (Fisher Scientific, Waltham, MA).
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4

Lymphocyte Isolation and Lipid Mediator Analysis

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Lymphocyte separation medium, aprotinin, dimethyl sulfoxide (DMSO) and solvents for HPLC and LC/MS were purchased from Thermo Fisher Scientific (Ottawa, Ontario, Canada). Dextran, adenosine deaminase, leupeptin and potassium phosphate were obtained from Sigma-Aldrich Canada (Oakville, Ontario, Canada). HBSS was purchased from Wisent Bioproducts (St-Bruno, Quebec, Canada). 19-OH-prostaglandin (PG) B2, PGB2, PGB2-D4 and 17-octadecynoic acid (17-ODYA) were purchased from Cayman Chemicals (Ann Arbor, Michigan, USA). PF-4708671 was obtained from Abcam (Cambridge, Massachusetts, USA) and LY2584702 from Selleckchem (Houston, Texas, USA). Thapsigargin was obtained from Tocris Bioscience (Ellisville. Missouri, USA). LTB4 was a generous gift from Dr Louis Flamand (Université Laval, Québec City, Canada). Recombinant CYP4F3A and the NADPH regenerating system were purchased from Corning (Corning, New York, USA). Protease and phosphatase inhibitor cocktail tablets were purchased from Roche (Laval, Quebec, Canada). Primary (anti-phospho-S6 #2211 and anti-S6 #2317) and secondary antibodies were obtained from Cell Signaling (Danvers, Massachusetts, USA). The enhanced chemiluminescent (ECL) substrate was obtained from Millipore Canada Ltd (Toronto, Ontario, Canada).
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5

Stimulation of Human PBMCs and Cytokine Measurement

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Human PBMCs were isolated from whole blood by using lymphocyte separation medium (Thermo Fisher Scientific, Waltham, MA). The freshly isolated PBMCs were cultured at 4 × 106/ml with GA (Teva Pharmaceutical Industries Ltd., Israel) at 50 µg/ml, LPS (InvivoGen, San Diego, CA) at 5 μg/ml, or CD3/CD28 mAbs (Biolegend, San Diego, CA) at 2 µg/ml for 3 days, and cytokine production was examined by ELISA (Biolegend, San Diego, CA). The remaining cells were frozen and stored in liquid nitrogen. The serum was isolated from the blood by using BD Vacutainer (Becton Dickinson, Franklin Lakes, NJ), and the serum concentration of cytokines was examined by LEGENDplex™ (Biolegend, San Diego, CA).
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6

Isolation of Lymphocyte Subsets

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For single-cell suspensions, thymus and spleen were crushed and filtered through 80μm stainless steel mesh (Sefar Ltd., UK) in fluorescence-activated cell sorting (FACS) buffer (phosphate-buffered saline (PBS) containing; 2% heat-inactivated fetal calf serum (FCS) (Life technologies); and 5 mM ethylenediaminetetraacetic acid (EDTA) (Life technologies)). For spleen, red blood cells were removed by gradient centrifiguation at 1600rpm for 25 min using 4 mL of Lymphocyte Separation Medium (Fischer). For IEL preparations, faecal material was flushed from lumen of small intestine with ice-cold PBS using a gavage needle. Fatty tissues, vasculature and Peyer’s patches were removed, followed by longitudinal opening and 60 min agitation in RPMI-1640 10% Newborn calf serum (NCS) (Life technologies) with 5 mM EDTA (Life technologies) at 37 °C. Cells were subsequently passed through an autoclaved column containing 0.7 g of nylon wool (Polysciences, USA), equilibrated with RPMI-1640 with 38 mM HEPES (Life technologies). IELs were enriched on a discontinuous Percoll gradient (40%/80% isotonic Percoll), before use and stained as described below.
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7

Isolation of Murine Splenocytes

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Mice were euthanized by CO2 narcosis followed by cervical dislocation. After euthanasia, spleens were removed, cleaned of fat and connective tissue and placed in cold RPMI buffered with 20 mM Hepes (ThermoFisher, Waltham MA). Cell suspensions were created by passing through a 70 um nylon screen and rinsing with additional RPMI. Splenocytes were pelleted at 900 × g for 6 minutes and suspended in cold RPMI. Splenocytes were then further purified by density gradient centrifugation utilizing Lymphocyte separation medium (ThermoFisher, Waltham MA). Cells were resuspended in serum-free RPMI 1640 supplemented with 20mM Hepes.
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8

Splenocyte Isolation from Murine Spleen

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Mice were euthanized by CO2 narcosis followed by cervical dislocation 24 hrs after SEB injection. After euthanasia, spleens were removed, cleaned of fat and connective tissue and placed in cold RPMI buffered with 20 mM Hepes (ThermoFisher, Waltham MA). Cell suspensions were created by passing through a 70 um stainless steel screen and rinsing with additional RPMI. Splenocytes were pelleted at 900 × g for 6 minutes and suspended in cold RPMI. Splenocytes were then further purified by density gradient centrifugation utilizing Lymphocyte separation medium (ThermoFisher, Waltham MA).
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9

PBMC Isolation and CD4+ T Cell Enrichment

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Apheresis leukoreduction collars, obtained from the Brigham and Women’s Hospital Crimson Core, were used to isolate peripheral blood mononuclear cell (PBMC) by Lymphocyte Separation Medium (ThermoFisher Scientific) density centrifugation. CD4+ T cells were isolated by negative selection using EasySep Human CD4+ T cell Enrichment Kit (StemCell Technologies). CD4+ T lymphocytes were cultured at a density of approximately 1 million cells/mL in RPMI-1640 (ThermoFisher Scientific) supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin.
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10

Isolation and characterization of leukemia cells

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Leukemia cell samples were isolated from patients’ peripheral blood using lymphocyte separation medium (Thermo Fisher Scientific) and incubated in suspension in the RPMI 1640 medium described above. Patient 1 had T-cell prolymphocytic leukemia (T-PLL), patient 2 had mixed phenotype acute leukemia and patient 3 had AML. Patient samples were collected after obtaining written informed consent, and all studies using these samples were performed under a protocol approved by the Institutional Review Board of the UT MD Anderson Cancer Center, in accordance with the Declaration of Helsinki.
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