Lympholyte h

Manufactured by Euroclone

Sourced in Italy

Lympholyte®-H is a density gradient medium designed for the separation and isolation of human lymphocytes from whole blood or buffy coat samples. It is a sterile, pyrogen-free solution that allows for the efficient recovery of viable lymphocytes with minimal contamination from other blood cell types.

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Lab products found in correlation

Ecl select western blotting detection reagent, by GE Healthcare (2 mentions) Anti mmp9 antibody, by Cell Signaling Technology (2 mentions) Sharpmass 6 mw marker, by Euroclone (2 mentions) Rpmi 1640, by Euroclone (2 mentions) Protran, by Cytiva (2 mentions) Triton x 100, by Merck Group (1 mentions) Acrylamide bis solution, by Bio-Rad (1 mentions) Ecl western blotting detection reagent, by GE Healthcare (1 mentions) Anti erk1 2 antibody, by Cell Signaling Technology (1 mentions) Anti p erk1 2 antibody, by Cell Signaling Technology (1 mentions) Amersham protran premium 0.45 m nitrocellulose, by GE Healthcare (1 mentions) Protease inhibitor cocktail 100, by Cell Signaling Technology (1 mentions) Dibutyryl camp, by Merck Group (1 mentions) Phosphatase inhibitor cocktail 100x, by Cell Signaling Technology (1 mentions) Raw264.7 cell line, by American Type Culture Collection (1 mentions) Fetal bovine serum (fbs), by Euroclone (1 mentions) Foetal bovine serum (fbs), by Euroclone (1 mentions) Penicillin, by Euroclone (1 mentions) Streptomycin, by Euroclone (1 mentions) L glutamine, by Euroclone (1 mentions)

5 protocols using lympholyte h

Isolation and Differentiation of Monocyte-Derived Macrophages

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Monocytes were isolated from buffy coats obtained from eight normal healthy donors provided by Immunohematology and Transfusion Medicine, University Hospital of Parma, Italy. Buffy coats, diluted 1:4 with PBS, were layered on 15 mL Lympholyte H (Euroclone, Milano, Italy) and centrifuged at 800× g for 20 min at 20 °C. PBMCs at the interface were collected and, after two washes in PBS, were suspended in complete growth medium (RPMI containing 10% endotoxin-free FBS) and seeded on plasticware. After a 30-min incubation at 37 °C, nonadherent cells were removed with three vigorous washes in PBS. Monocyte derived macrophages (MDMs) were obtained by incubating monocytes in complete growth medium added with 50 ng/mL of recombinant human Granulocyte Mϕ-Colony-Stimulating Factor (GM-CSF, Vinci-Biochem, Firenze, Italy) for up to 6 d. The study was approved by the local ethical committee (460/2021/TESS/UNIPR).
Human Umbilical Vein Endothelial Cells (HUVEC) were purchased from Cell Applications, Inc. (Merck, Milano, Italy) and routinely grown according to the manufacturer’s instructions.

Barilli A., Visigalli R., Ferrari F., Recchia Luciani G., Soli M., Dall’Asta V, & Rotoli B.M. (2022). The JAK1/2 Inhibitor Baricitinib Mitigates the Spike-Induced Inflammatory Response of Immune and Endothelial Cells In Vitro. Biomedicines, 10(9), 2324.

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Plasma Separation and DNA Extraction

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Blood samples were obtained by venepuncture from patients using lithium-heparin anti-coagulant and transported to the laboratory at 4°C. In the laboratory, 20 ml of blood were slow pipetted to 20 ml of Lympholyte®-H, sterile liquid (EuroClone) and centrifuged at 400 xg for 40 minutes without brake to obtain blood stratification. Approximately 6 mL of plasma were transferred in a 15 mL centrifuge tube and stored at -80°C. One ml of each sample was used for subsequent DNA extraction.

Cocuzza C.E., Martinelli M., Sina F., Piana A., Sotgiu G., Dell’Anna T, & Musumeci R. (2017). Human papillomavirus DNA detection in plasma and cervical samples of women with a recent history of low grade or precancerous cervical dysplasia. PLoS ONE, 12(11), e0188592.

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Investigation of MMP9 and ERK1/2 Signaling

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RPMI 1640, Lympholyte^®-H, and pre-stained protein SHARPMASS VI MW marker were purchased from Euroclone SpA (Milan, Italy). Anti-MMP9 antibody, anti-P-ERK 1/2 antibody, anti-ERK1/2 antibody, horseradish peroxidase (HRP)-linked anti-rabbit and anti-mouse secondary antibodies, protease inhibitor cocktail (100×), and phosphatase inhibitor cocktail (100X) were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-IkBα and anti-β-actin antibodies were obtained from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Dibutyryl-cAMP, the potentiator ivacaftor (VX770), gelatin, Triton X-100, and BRIJ^®35 Detergent Calbiochem^® were purchased from Sigma-Aldrich (Milan, Italy). ECL Select™ Western Blotting Detection Reagent, ECL Western Blotting Detection Reagent, and Amersham™ Protran^® Premium 0.45-µm nitrocellulose were obtained from GE Healthcare (Chicago, IL, USA). Brillant Blue R-250 and Acrylamide/Bis Solution were obtained from Bio-Rad Laboratories Srl (Segrate, MI, Italy).

Capraro M., Pedrazzi M., De Tullio R., Manfredi M., Cresta F., Castellani C, & Averna M. (2023). Modulation of Plasmatic Matrix Metalloprotease 9: A Promising New Tool for Understanding the Variable Clinical Responses of Patients with Cystic Fibrosis to Cystic Fibrosis Transmembrane Conductance Regulator Modulators. International Journal of Molecular Sciences, 24(17), 13384.

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Macrophage Cell Culture and Isolation

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The murine macrophage RAW264.7 cell line was purchased from ATCC. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum (FBS, Euroclone), 2 mM L‐glutamine (Euroclone), 100 U/ml penicillin and 100 μg/ml streptomycin (Euroclone).
Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy donors using Ficoll gradient centrifugation (Lympholyte‐H, Euroclone). After their viability was checked by Trypan blue exclusion test at the optical microscope, PBMCs were cultured in RPMI 1640 supplemented with 15 mM HEPES Buffer, 100 IU/ml penicillin, 0.1 mg/ml streptomycin, 2 mM L‐glutamine and 10% heat‐inactivated foetal bovine serum (Euroclone).
Human monocytes were isolated and purified from PBMCs of buffy coats obtained from healthy donors. The procedures adopted in this study were in agreement with the Helsinki Declaration and were approved by the Ethics Committee of the University Hospital of Palermo, Palermo, Italy (ethical protocol code N° 03/2019 and N° 01/2022). After isolation from PBMCs, using Pan Monocyte Kit on MACS Separator (Miltenyi Biotec) according to protocol, monocytes were cultured at a concentration of 2 × 10⁵/ml in presence of macrophage colony‐stimulating factor (M‐CSF; 20 ng/ml) for 1 week to induce differentiation into macrophages.

Raimondo S., Urzì O., Meraviglia S., Di Simone M., Corsale A.M., Rabienezhad Ganji N., Palumbo Piccionello A., Polito G., Lo Presti E., Dieli F., Conigliaro A, & Alessandro R. (2022). Anti‐inflammatory properties of lemon‐derived extracellular vesicles are achieved through the inhibition of ERK/NF‐κB signalling pathways. Journal of Cellular and Molecular Medicine, 26(15), 4195-4209.

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CFTR Inhibitor and Potentiator Assay

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RPMI 1640, fetal bovine serum (FBS), penicillin–streptomycin solution 100X, L-glutamine 100X 200 mM, Lympholyte^®-H, and prestained protein SHARPMASS VI MW marker were purchased from Euroclone SpA (Milan, Italy); anti-MMP9 antibody, horseradish peroxidase (HRP)-linked anti-rabbit secondary antibody, protease inhibitor cocktail (100X), and phosphatease inhibitor cocktail (100X) were obtained from Cell Signaling Technology (Danvers, MA, USA); dibutyryl-cAMP, isopropyl β-D-1-thiogalactopyranoside (IPTG), yeast extract, tryptone, PGex6P1, 6,7-Dihydro-7,9-dimethyl-6-(5-methyl-2-furanyl)-11-phenylpyrimido (4′,5′,3,4) pyrrolo (1,2-a) quinoxaline –8,10 (5H,9)-dione (PPQ-102), a reversible and voltage-independent CFTR inhibitor, and the potentiator Ivacaftor (VX770) were purchased from Sigma-Aldrich (Milan, Italy); GSH-sepharose™, ECL Select™ Western Blotting Detection Reagent, and Amersham ™ Protran^® Premium 0.45-μm nitrocellulose were obtained from GE Healthcare (Chicago, IL, USA); Monocytes Isolation Kit II was purchased from Miltenyi Biotec Srl (Bologna, Italy); pEYFP-C1 plasmid was obtained from Clontech Laboratories (Mountain View, CA, USA); QuickChange site-directed mutagenesis kit was from Stratagene (San Diego, CA, USA); BamHI and EcoRI restriction enzymes by Fermentas were purchased from Life Technologies Italia (Monza, Italy).

Pedrazzi M., Vercellone S., Barberis E., Capraro M., De Tullio R., Cresta F., Casciaro R., Castellani C., Patrone M., Marengo E., Lecca P., Melotti P., Sorio C., Manfredi M, & Averna M. (2021). Identification of Potential Leukocyte Biomarkers Related to Drug Recovery of CFTR: Clinical Applications in Cystic Fibrosis. International Journal of Molecular Sciences, 22(8), 3928.

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