Lymphoprep

Manufactured by Fresenius

Sourced in Norway, Germany

Lymphoprep is a sterile, endotoxin-tested, density gradient medium used for the isolation of mononuclear cells, such as lymphocytes, from whole blood or other cellular suspensions. It is a ready-to-use solution that enables the separation of cell types based on their different buoyant densities.

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Lab products found in correlation

Close spelling variants of this product

Lymphoprep density separation (8 citations) Isopaque-Ficoll (Lymphoprep; (4 citations)

56 protocols using lymphoprep

Isolation and Characterization of B-ALL Cells

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B-ALL patient samples were obtained after informed consent following the tenets of the Declaration of Helsinki. The study was approved by the Ethical Committee board of the University of Padova, the Padova Academic Hospital and the Italian Association of Pediatric Onco-Hematology (AIEOP). Diagnosis was made according to standard cytomorphology, cytochemistry and immunophenotypic criteria [46 ]. All analyzed B-ALL samples were obtained at the time of diagnosis before treatment, after Lymphoprep (Fresenius KABI, Norge AS) separation of mononuclear cells. The percentage of CD19⁺ cells ranged from 80% to 95%.
Peripheral blood mononuclear cells (PBMC) and bone marrow cells from healthy donors were obtained by separation on Lymphoprep (Fresenius KABI, Norge AS) gradient. After extensive washing, cells were resuspended (1.0 × 10⁶ cells/ml) in RPMI1640 with 10% fetal bovine serum and incubated overnight in 96-well tissue culture microtiter plate. For cytotoxicity evaluations in proliferating PBMC cultures, non-adherent cells were resuspended in growth medium, containing 2.5 μg/ml phytohematoglutinin (PHA) (Irvine Scientific). To isolate B-lymphocyte, PBMC obtained by Lymphoprep (Fresenius KABI, Norge AS) separation were labeled with anti-CD19-APC (BD Biosciences, Italy) and collected by cell sorting.

Bortolozzi R., Viola G., Porcù E., Consolaro F., Marzano C., Pellei M., Gandin V, & Basso G. (2014). A novel copper(I) complex induces ER-stress-mediated apoptosis and sensitizes B-acute lymphoblastic leukemia cells to chemotherapeutic agents. Oncotarget, 5(15), 5978-5991.

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PBMC Isolation from Blood Samples

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We drew 20-ml blood samples from each patient. Blood was overlaid onto Lymphoprep (Lymphoprep; Fresenius Kabi, Oslo, Norway). PBMCs were isolated through a centrifugation procedure.

Brumnjak S.V., Rakovac I., Kinkela D.P., Bukal K., Sestan B., Tulic V., Janjetic E.V, & Tokmadzic V.S. (2018). Postoperative Regional Analgesia Is Effective in Preserving Perforin-Expressing Lymphocytes in Patients After Total Knee Replacement. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 24, 5320-5328.

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Phenotyping of Th cell subsets

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Peripheral blood mononuclear cells (PBMCs) were isolated from EDTA-treated whole blood samples by density gradient centrifugation using Lymphoprep ™ (Fresenius Kabi, Oslo, Norway).
Peripheral blood lymphocytes (PBL) were isolated and stained with the following fluorochrome-conjugated monoclonal antibodies: CD4-APC-H7, CXCR3-PE, CCR6-BB515, PD-1-PE-Cy7 and ICOS-BV450 (all from BD biosciences, San José, CA, USA) (Mallett et al., 2019 (link); Mousset et al., 2019 (link)). Acquisition of PBLs was performed with a FACS CANTO II flow cytometry (BDbiosciences) and analyzed with Flow-Jo software v10.6.2. Gating strategy is shown (Figure S1).
Cells CXCR3+/CCR6- gated from CD4 were considered Th1 cells, CXCR3-/CCR6+ cells were considered as Th17 and CXCR3-/CCR6- as Th2. Regarding the activation grade, cells PD-1-/ICOS- were considered as quiescent Th cells, PD-1-/ICOS+ cells as early-activated cells, PD-1+/ICOS+ as late activation markers and PD-1+/ICOS- as exhausted or senescent cells (Mallett et al., 2019 (link)).

Gil-Etayo F.J., Suàrez-Fernández P., Cabrera-Marante O., Arroyo D., Garcinuño S., Naranjo L., Pleguezuelo D.E., Allende L.M., Mancebo E., Lalueza A., Díaz-Simón R., Paz-Artal E, & Serrano A. (2021). T-Helper Cell Subset Response Is a Determining Factor in COVID-19 Progression. Frontiers in Cellular and Infection Microbiology, 11, 624483.

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Isolation and Cytotoxicity Evaluation of PBLs

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PBLs were obtained from human peripheral blood (leucocyte rich plasma-buffy coats) from
healthy volunteers using the Lymphoprep (Fresenius KABI Norge AS) gradient density
centrifugation.
Buffy coats were obtained from the Blood Transfusion Service, Azienda Ospedaliera of
Padova and provided at this institution for research purposes. Therefore, no further
informed consent was needed. In addition, buffy coats were provided without identifiers.
The experimental procedures were carried out in strict accordance with approved
guidelines.
After extensive washing, cells were resuspended (1.0 × 10⁶ cells/mL) in
RPMI-1640 with 10% fetal bovine serum and incubated overnight. For cytotoxicity
evaluations in proliferating PBL cultures, nonadherent cells were resuspended at 5
× 10⁵ cells/mL in growth medium, containing 2.5 μg/mL PHA (Irvine
Scientific). Different concentrations of the test compounds were added, and viability
was determined 72 h later by the MTT test. For cytotoxicity evaluations in resting PBL
cultures, nonadherent cells were resuspended (5 × 10⁵ cells/mL) and
treated for 72 h with the test compounds.

Spanò V., Rocca R., Barreca M., Giallombardo D., Montalbano A., Carbone A., Raimondi M.V., Gaudio E., Bortolozzi R., Bai R., Tassone P., Alcaro S., Hamel E., Viola G., Bertoni F, & Barraja P. (2020). Pyrrolo[2′,3′:3,4]cyclohepta[1,2-d][1,2]oxazoles, a New Class of Antimitotic Agents Active against Multiple Malignant Cell Types. Journal of Medicinal Chemistry, 63(20), 12023-12042.

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Isolation and Expansion of Bone Marrow-Derived Mesenchymal Cells

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40–50 ml of bone marrow was aspirated from the iliac crest of one healthy voluntary donor, and the mononuclear cells were isolated from the aspirate using density gradient centrifugation (Lymphoprep, Fresenius Kabi, Oslo, Norway). The cells were seeded in 175 cm² flasks (Nunc, Roskilde, Denmark) and cultured in Dulbecco’s Modified Eagle Medium:Nutrient Mixture F-12 containing GlutaMAX (DMEM/F-12 GlutaMAX, Gibco, Paisley, UK), supplemented with 10% pooled human platelet lysate plasma (PLP), 1% penicillin/streptomycin and 1.5 µg/ml amphotericin B. After 24–48 hours, non-adherent cells were removed by medium change and the remaining cells were expanded using the medium described above, but without amphotericin B.

Olderøy M.Ø., Lilledahl M.B., Beckwith M.S., Melvik J.E., Reinholt F., Sikorski P, & Brinchmann J.E. (2014). Biochemical and Structural Characterization of Neocartilage Formed by Mesenchymal Stem Cells in Alginate Hydrogels. PLoS ONE, 9(3), e91662.

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Single-cell analysis of nasal and blood samples

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Polyps were sampled from the nasal cavity, middle and superior meatus, and ethmoid sinuses from CRSwNP, AFRS, and EMCRS patients. Matched heparinized venous PB samples were obtained from every patient during sinus surgery for comparative analysis. Only PB samples were studied in ARFA and HCs.
Single cell preparation of fresh sinus tissue samples for flow cytometry was performed as previously described, and experiments were performed within a few hours.^{14 (link)} Briefly, whole pieces of tissue were washed to remove macroscopic blood and were minced to <2-mm pieces and passed through an 80-μm nylon mesh (BD Biosciences, San Jose, CA) to isolate single cells without the use of digestive enzymes. Lymphocyte viability was determined by propidium iodide staining and was >92% and viable cells were used for flow cytometry.
PB mononuclear cells (PBMCs) for proliferation and flow cytometry studies were prepared by standard density gradient centrifugation (Lymphoprep; Fresenius Kabi, Bad Homburg, Germany) of heparinized venous PB samples.

Pant H, & Macardle P. (2014). CD8+ T cells implicated in the pathogenesis of allergic fungal rhinosinusitis. Allergy & Rhinology, 5(3), e146-e156.

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Isolation and Culture of Myeloma and Leukemia Cell Lines

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NCI-H929, U266B, JIM3 and OPM2 human MM cell lines, KG-1 (an acute myeloid leukemia cell line) and HS-5 (fibroblast-like) and HS-27 (epithelial-like) BMSCs were obtained from the American Type Culture Collection and cultured in RPMI-1640 (Sigma-Aldrich) supplemented with 10% fetal calf serum (FCS; Gibco). All cells were routinely tested for mycoplasma and were free from infection. Isolation of peripheral blood mononuclear cells (PBMC): healthy donor blood was obtained from leukocyte apheresis cones supplied by the National Health Service Blood and Transplant unit. PBMC were isolated using Lymphoprep (Fresenius-Kabi) and seeded at 2×10⁶ cells/mL in RPMI-1640 supplemented with 10% FCS.

Müller L.M., Migneco G., Scott G.B., Down J., King S., Askar B., Jennings V., Oyajobi B., Scott K., West E., Ralph C., Samson A., Ilett E.J., Muthana M., Coffey M., Melcher A., Parrish C., Cook G., Lawson M, & Errington-Mais F. (2021). Reovirus-induced cell-mediated immunity for the treatment of multiple myeloma within the resistant bone marrow niche. Journal for Immunotherapy of Cancer, 9(3), e001803.

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Isolation and Preservation of PBMCs

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Heparinised blood and sera were collected from each patient at diagnosis and throughout NC, after 12 and 24 weeks of treatment. Peripheral blood mononuclear cells (PBMCs) were freshly isolated from heparinised blood of patients by Ficoll-Hypaque gradient (Lymphoprep, Fresenius Kabi Norge Halden, Norway) using standard procedures and viably frozen at -180°C until use. Serum samples were obtained with blood centrifugation at 2,100 rpm and maintained at -80°C.

Miolo G., Muraro E., Martorelli D., Lombardi D., Scalone S., Spazzapan S., Massarut S., Perin T., Viel E., Comaro E., Talamini R., Bidoli E., Turchet E., Crivellari D, & Dolcetti R. (2014). Anthracycline-free neoadjuvant therapy induces pathological complete responses by exploiting immune proficiency in HER2+ breast cancer patients. BMC Cancer, 14, 954.

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Isolation and Enrichment of CD8+ T Cells for RA Analysis

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Blood samples were collected from 15 RA patients and eight healthy controls for microarray analysis, and 25 RA patients for the in vitro stimulation with nicotine. Peripheral blood mononuclear cells (PBMC) were isolated through gradient centrifugation using Lymphoprep (Fresenius Kabi, Oslo, Norway). CD8⁺ T cells were enriched in the PBMC using Stemcell negative selection human CD8⁺ T cell enrichment kit according to the manufacturer's instructions. The purity of CD8⁺ cells were 81–92%, as determined by flow cytometry.

Wasén C., Ospelt C., Camponeschi A., Erlandsson M.C., Andersson K.M., Silfverswärd S.T., Gay S, & Bokarewa M.I. (2020). Nicotine Changes the microRNA Profile to Regulate the FOXO Memory Program of CD8+ T Cells in Rheumatoid Arthritis. Frontiers in Immunology, 11, 1474.

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FACS analysis of GR expression and binding in leukocytes

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GR expression and binding capacity in different leucocyte subsets were assessed using a FACS CantoII (BD Biosciences) equipped with Diva software (BD Biosciences). For each sample, at least 10,000 events were collected in the lymphocyte gate. Fluorescence was compensated using BD Anti-mouse CompBeads (BD Biosciences). For control of day-to-day variations of the assay, leucocytes from one healthy blood donor were included in each analysis. This internal standard was prepared sterile at one time as follows. Peripheral blood mononuclear cells (PBMCs) were separated using Lymphoprep (Fresenius Kabi, Oslo, Norway), washed in ice cold sterile PBS, and frozen in heat inactivated fetal calf serum with 15 % DMSO at a rate of −1 °C/min using a Nalgene® Mr. Frosty™ Freezing Container (Thermo Scientific, Waltham, MA, USA). For each analysis, a vial of the internal standard was defrosted on ice and washed in FACS buffer followed by surface and intracellular staining together with each patient sample. Data was further processed using FlowJo software (Tree Star, Inc., Ashland, VA, USA).

Bergquist M., Lindholm C., Strinnholm M., Hedenstierna G, & Rylander C. (2015). Impairment of neutrophilic glucocorticoid receptor function in patients treated with steroids for septic shock. Intensive Care Medicine Experimental, 3, 23.

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