Neutralization assays with serum IgGs against the 12-strain global virus panel, were performed in 96-well plates following published protocols9 (link),90 (link). To this end, 12 HIV-1 pseudovirus strains were each mixed with 1:2 serial dilutions of purified IgG (1 mg/ml starting concentration, 8 dilutions) and incubated for 1 h at 37 °C. TZM-bl cells (RRID:CVCL_B478; ordered from the HIV Reagent Program, Cat-No. ARP-8129) were added (104 per well) in growth medium (DMEM, Gibco; 10% heat-inactivated FBS, Sigma-Aldrich; 2 mM L-glutamine, Thermo Fisher; 1 mM sodium pyruvate, Gibco; 50 µg/ml gentamicin, Sigma-Aldrich; 25 mM HEPES, Biochrom) with DEAE-dextran at a final concentration of 10 µg/ml and incubated for 2 days. Equal amounts of Luciferin-containing lysis buffer (10 mM MgCl2, 0.3 mM ATP, 0.5 mM Coenzyme A, 17 mM IGEPAL (all Sigma-Aldrich), and 1 mM D-Luciferin (GoldBio) in 200 mM Tris-HCL pH 7.8) was added and after 2 min incubation samples were resuspended and luminescence was measured with a luminometer (Berthold TriStar2 LB942). For IC50 determination, the background signal (non-infected TZM-bl cells) was subtracted and IgG concentrations resulting in a 50% RLU reduction compared to untreated virus control wells were determined by using murine leukemia virus (MuLV)-pseudotyped virus as a control for unspecific activity. All samples were tested in duplicates.
Hepes
Manufactured by Harvard Bioscience
Sourced in Germany
HEPES is a buffering agent commonly used in cell culture and biochemical applications. It helps maintain a stable pH environment for biological processes. HEPES is a zwitterionic organic compound that effectively buffers solutions in the physiological pH range.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Harvard Bioscience (13 mentions)
Penicillin, by Harvard Bioscience (10 mentions)
Streptomycin, by Harvard Bioscience (9 mentions)
L glutamine, by Harvard Bioscience (8 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (7 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (7 mentions)
Sodium pyruvate, by Harvard Bioscience (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
β mercaptoethanol, by Merck Group (5 mentions)
Non essential amino acids (neaa), by Harvard Bioscience (5 mentions)
L glutamine, by Thermo Fisher Scientific (5 mentions)
Fetal calf serum (fcs), by Harvard Bioscience (5 mentions)
Streptomycin, by Thermo Fisher Scientific (5 mentions)
Fetal calf serum (fcs), by Merck Group (5 mentions)
Gentamicin, by Merck Group (4 mentions)
Penicillin, by Merck Group (4 mentions)
Glutamine, by Harvard Bioscience (4 mentions)
Fetal bovine serum (fbs), by Harvard Bioscience (4 mentions)
Rpmi 1640, by Thermo Fisher Scientific (4 mentions)
Penicillin, by Thermo Fisher Scientific (4 mentions)
60 protocols using hepes
1
SARS-CoV-2 Neutralization Assay with IgGs
2
Derivation of human macrophages
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For hMDMs (human
monocyte-derived macrophages), peripheral blood mononuclear cells
(PBMCs) were isolated from human buffy coat via high density gradient
centrifugation (Ficoll-Paque Plus; GE Healthcare, Munich). Monocytes
were then purified from the PBMCs through adherence. Then, the cells
were stimulated with granulocyte macrophage colony-stimulating factor
(GM-CSF) (10 ng/mL; MiltenyiBiotec, BergischGladbach) for 6 days at
37 °C and 5% CO2 in RPMI 1640 medium (GIBCO, Invitrogen,
Munich) supplemented with 2 mMl -glutamine (PAN Biotech,
Aidenbach), 10 mM HEPES (Biochrom, GmbH, Berlin), and penicillin/streptomycin
(100 U mL–1/100 μg mL–1 (Biochrom,
GmbH, Berlin).
For the THP-1 cell line (ECACC, Porton Down,
UK, 88081201), human monocytic cell line derived from an acute monocytic
leukemia patient was differentiated to macrophage-like cells in the
cell culture medium. Mercaptoethanol (0.05 mM; Sigma, Merck KGaA,
Darmstadt), 10% FCS (Biochrom, GmbH, Berlin), and phorbol 12-myristate
13-acetate (PMA) (1 μg/mL; Sigma, Merck KGaA, Darmstadt) were
used as supplemented at 36 °C/5%CO2 overnight.
monocyte-derived macrophages), peripheral blood mononuclear cells
(PBMCs) were isolated from human buffy coat via high density gradient
centrifugation (Ficoll-Paque Plus; GE Healthcare, Munich). Monocytes
were then purified from the PBMCs through adherence. Then, the cells
were stimulated with granulocyte macrophage colony-stimulating factor
(GM-CSF) (10 ng/mL; MiltenyiBiotec, BergischGladbach) for 6 days at
37 °C and 5% CO2 in RPMI 1640 medium (GIBCO, Invitrogen,
Munich) supplemented with 2 mM
Aidenbach), 10 mM HEPES (Biochrom, GmbH, Berlin), and penicillin/streptomycin
(100 U mL–1/100 μg mL–1 (Biochrom,
GmbH, Berlin).
For the THP-1 cell line (ECACC, Porton Down,
UK, 88081201), human monocytic cell line derived from an acute monocytic
leukemia patient was differentiated to macrophage-like cells in the
cell culture medium. Mercaptoethanol (0.05 mM; Sigma, Merck KGaA,
Darmstadt), 10% FCS (Biochrom, GmbH, Berlin), and phorbol 12-myristate
13-acetate (PMA) (1 μg/mL; Sigma, Merck KGaA, Darmstadt) were
used as supplemented at 36 °C/5%CO2 overnight.
3
FPR2/ALX-Expressing HL60 Cell Calcium Flux
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HL60 cells stably transfected with human FPR2/ALX have been recently described (5 (link)). These cells were grown in RPMI medium (Biochrom) supplemented with 10% FCS (Sigma-Aldrich), 20 mM HEPES (Biochrom), penicillin (100 units/ml), streptomycin (100 µg/ml) (Gibco), 1× GlutaMAX (Gibco), and G418 (Biochrom) at a final concentration of 1 mg/ml. Calcium fluxes were analyzed by stimulating cells loaded with Fluo-3-AM (Molecular Probes), and the fluorescence was monitored with a FACSCalibur flow cytometer (Becton, Dickinson), as recently described (39 (link)).
4
Cell Culture in Supplemented RPMI 1640
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Cells were cultured in RPMI 1640 medium (Life Technologies) supplemented with penicillin (50 U/ml), streptomycin (50 U/ml), HEPES (25 mM), sodium pyruvate (1 mM), β-mercaptoethanol (50 μM) (all purchased from Biochrom) containing 10 % fetal calf serum (Sigma-Aldrich), at 37°C, 5 % CO2 in 96U bottom plates (Corning).
5
BDNF Expression in hMSCs via Lentiviral Transduction
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The expression of human BDNF (entire coding sequence including signal peptide: Warnecke et al., 2012 (link)) was under the control of a spleen focus-forming virus (SFFV) promoter in a lentiviral vector that also mediated red fluorescence using the marker protein tdTomato (red). Subsequently, after lentivirus production, hMSCs from one selected donor were seeded at 3,000 cells/cm2, passage 4 or 5, and were infected with the BDNF-lentivirus including 8 μg/ml polybrene. In order to subsequently downgrade the cells to S1 level, the cells were cultured and expanded for 11 days before being harvested with trypsin/EDTA solution. The medium used for expansion of MSCs (‘MSC medium’) was Dulbecco’s Modified Eagle’s Medium (1 g/l glucose, Biochrom, FG0415) supplemented with 10% (v/v) fetal calf serum (FCS, not heat-inactivated, Thermo Fisher Scientific, Schwerte, Germany, ‘HyClone’, SV30160.03), 25 mM HEPES (Biochrom, Berlin, Germany), 1% (100 U/ml/100 μg/ml) penicillin/streptomycin (Biochrom, Berlin, Germany) and 2 ng/ml human recombinant FGF 2 (from Escherichia coli, PeproTech, Hamburg, Germany).
6
Cultivation and Isolation of Hepatocytes
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HepG2 hepatocytes (ACC 180, DSMZ, Germany) were cultivated in DMEM containing HEPES (25 mM), FCS (10%), penicillin/streptomycin (1%) and l -glutamine (2 mM, all Biochrom, Germany).
For cultivation of HepG2-3A4 (source BTU Cottbus-Senftenberg, Germany)30 (link) the medium of HepG2 was modified by supplementing 3 µg/ml blasticidin (AppliChem, Germany). Both cell lines were cultured at 37 °C and 5% CO2. For subculture, the cells were rinsed twice with PBS and incubated with 0.25% Trypsin/EDTA (Biochrom, Germany) for 7 min at 37 °C.
Primary mouse hepatocytes were isolated from C57/BL6 mice as described previously32 (link). Briefly, the liver was first perfused with calcium- and magnesium-free Hank's balanced salt solution containing 1 mM EDTA followed by a perfusion with Hank's balanced salt solution containing 0.016 mg/ml Liberase (Roche, Germany). Hepatocytes were separated from non-parenchymal cells and debris by density centrifugation with 58% Percoll in phosphate buffered saline.
The principles of laboratory animal care were followed. Treatment of the animals followed the German animal protection laws and was performed with permission of the state animal welfare committee (LUGV Brandenburg, 2347-A-16-2-2017).
For cultivation of HepG2-3A4 (source BTU Cottbus-Senftenberg, Germany)30 (link) the medium of HepG2 was modified by supplementing 3 µg/ml blasticidin (AppliChem, Germany). Both cell lines were cultured at 37 °C and 5% CO2. For subculture, the cells were rinsed twice with PBS and incubated with 0.25% Trypsin/EDTA (Biochrom, Germany) for 7 min at 37 °C.
Primary mouse hepatocytes were isolated from C57/BL6 mice as described previously32 (link). Briefly, the liver was first perfused with calcium- and magnesium-free Hank's balanced salt solution containing 1 mM EDTA followed by a perfusion with Hank's balanced salt solution containing 0.016 mg/ml Liberase (Roche, Germany). Hepatocytes were separated from non-parenchymal cells and debris by density centrifugation with 58% Percoll in phosphate buffered saline.
The principles of laboratory animal care were followed. Treatment of the animals followed the German animal protection laws and was performed with permission of the state animal welfare committee (LUGV Brandenburg, 2347-A-16-2-2017).
7
Generation of Stable HeLa Cell Lines
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For generation of stable cell lines, transfected HeLa cells were selected for stable neomycine resistance by growth in the presence of 0.8 mg/ml G418 (Calbiochem 345810). Untransfected HeLa cells and the stable HeLa cell lines (CoxVIIc-, CoxVIIIa-, CoxVIIIa-Link-, CoxIV- and mt-sEcGFP) were cultured in Minimal Essential Medium with Earle’s salts (MEM, PAA Lab GmbH, E15–888) with 5.6 mM glucose, 2 mM stable glutamine and sodium bicarbonate, supplemented with 10% (v/v) fetal bovine serum (FBS) superior (Biochrom AG), 1% MEM nonessential amino acids (NEAA, Biochrom AG) and 1% 4−(2−hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES, PAA Lab GmbH) at 37 °C with 5% CO2. Stable HeLa cell lines were kept in 0.8 mg/ml G418 in addition. Cells were split 2–3 times a week using Trypsin/EDTA (Biochrom), supplemented with HEPES (PAA), sodium bicarbonate (PAA), penicillin/streptomycin (Biochrom) and PBS (Biochrom). For nanoenvironment determination by fluorescence lifetime measurements, sEcGFP cell lines were seeded onto glass coverslips in 3.5 cm cell culture dishes. Imaging was performed 2 days after seeding.
8
Isolation and Differentiation of Human Macrophages
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Buffy coat was obtained via the blood bank of Deutsches Rotes Kreuz (Springe, Germany) from pooled samples of voluntary, anonymous blood donors who gave informed consent. Lymphocytes were prepared from buffy coat by Ficoll-Hypaque density gradient centrifugation57 . Buffy coat was diluted in PBS (ratio 1:3) and Ficoll-Hypaque was added, following centrifugation for 20 min at 800×g. Afterwards, 5 × 108 cells were cultured in Roswell Park Memorial Institute (RPMI) medium containing 5.5 g × l−1 NaCl, 5.0 mg × l−1 phenol red, 2.0 g × l−1 NaHCO3, 25 mM HEPES, 4 mM stable glutamine without sodium pyruvate (Biochrom), 100 units × ml−1 penicillin, 100 µg × ml−1 streptomycin, 2.5 ng × ml−1 GM-CSF (granulocyte-macrophage colony-stimulating factor) and supplemented with 10% human plasma. After o/n incubation at 37 °C in an atmosphere of 5% CO2 and 90% humidity, non-adherent cells were removed, and adherent cells were cryo-preserved in inactivated fetal calf serum (iFCS) and DMSO. Next, cell suspensions were thawed, seeded, and maintained in RPMI-1640 (Biochrom), supplemented with 20% FCS and 2.5 ng × ml−1 GM-CSF (Peprotech) to differentiate monocytes into macrophages. After 5–7 days, the purity of the macrophage population was checked by staining with FITC anti-human CD14 antibody (BioLegend) and FC analyses. The macrophages were used for infection by STM strains.
9
Calu-3 Cell-Based Permeability Assay
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OP was kindly provided by the Nile Co. for Pharmaceuticals and Chemical Industries, Cairo, Egypt. Trehalose (coarse grade) was provided by Cargill Deutschland GmbH (Krefeld, Germany). Mannitol 60 (coarse grade) and glucose monohydrate (coarse grade) were provided from Roquette, Ludwigshafen, Germany. Lactose inhalation fine grades were of two types; Respitose® ML006 was kindly provided by DMV-Fronterra Excipients, Nörten-Hardenberg, Germany, while Lactohale® LH 300 was kindly provided by Friesland Foods Domo, Borculo, The Netherlands. Calu-3 (HTB-55) cells were purchased from the American type culture collection (ATCC, Manassas, VA, USA). MTT (3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide), Hank’s buffered salt solution (HBSS), HEPES (4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid) buffer solution, minimum essential medium (MEM) with Earle’s salt, fetal bovine serum, penicillin-streptomycin, nonessential amino acids, and sodium pyruvate were all supplied from Biochrom AG, Berlin, Germany. All reagents (Brij35, ethanol, glycerol, sodium dodecyl sulfate (SDS), dimethylformamide (DMF), trypsin, and Ethylenediaminetetraacetic acid (EDTA) used were of analytical grade and supplied by Merck KGaA (Darmstadt, Germany).
10
Isolation of Peripheral Blood Mononuclear Cells
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Twenty mL blood containing 75 units per mL sodium-heparin (SIGMA Taufkirchen, Germany) was layered over 20 mL lymphocyte separation medium (PAA, Pasching, Austria) and centrifuged at 1500 × g for 30 min at RT. The PBMC were removed, washed three times with Hank’s buffer without calcium and magnesium, re-suspended in RPMI 1640 medium with glutamine, 10% foetal calf serum (FCS), 10 mM HEPES and 1% Penicillin/Streptomycin (all Biochrom, Berlin, Germany) and adjusted to 2 x 106 PBMC per mL.
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