Cell culturing: Three different cell lines were used: HepG2, Calu-3 and Raw264.7 (ATCC, Manassas, VA, USA). HepG2 cells were cultured in flat-bottomed 96-well MicroWell™ plates (NUNC, Roskilde, DK) at a seeding density of 10,000 cells/well in EMEM supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL), L-glutamine (2 mM), sodium pyruvate (1 mM), non-essential amino acids (10 mg/mL), and fetal bovine serum (100 mg/mL) under standard culturing conditions (37 °C, 5% CO2, humidified air). Calu-3 cells were cultured in collagen-coated flat-bottomed 96-well MicroWell™ plates (NUNC) at a seeding density of 6,000 cells/well in DMEM supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL), L-glutamine (2 mM), and fetal bovine serum (100 mg/mL) under standard culturing conditions (37 °C, 5% CO2, humidified air). RAW 264.7 cells were cultured in flat-bottomed 96-well MicroWell™ plates (NUNC) at a seeding density of 6,000 cells/well in DMEM supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL), L-glutamine (2 mM), and fetal bovine serum (100 mg/mL) under standard culturing conditions (37 °C, 5% CO2, humidified air). Cells were cultured until 80-90% confluency was reached, and then washed twice with DMEM prior to use.
Microwell plate
Manufactured by Thermo Fisher Scientific
Sourced in Denmark
Microwell plates are a type of laboratory equipment used for various applications in life science research and diagnostics. They consist of an array of small, individual wells arranged in a grid-like format. Microwell plates are designed to hold small volumes of liquids, samples, or reagents, allowing for multiple experiments or tests to be conducted simultaneously.
Automatically generated - may contain errors
Lab products found in correlation
Silver stain kit, by Bio-Rad (3 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (3 mentions)
Monoclonal anti cd63 antibody clone ts63, by Abcam (3 mentions)
Vectastain elite abc kit, by Vector Laboratories (3 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (3 mentions)
Biotinylated goat anti mouse igg, by Vector Laboratories (2 mentions)
Biotinylated goat anti galectin 3 gal 3 antibodies, by R&D Systems (2 mentions)
Methyl alpha d mannopyranoside, by Merck Group (2 mentions)
3 3 5 5 tetramethylbenzidine tmb bovine serum albumin, by Merck Group (2 mentions)
Sds page molecular mass standards broad range, by Bio-Rad (2 mentions)
Automated plate reader, by Molecular Devices (1 mentions)
Peroxidase conjugated goat anti human igg, by Merck Group (1 mentions)
Raw264, by American Type Culture Collection (1 mentions)
Hepg2, by Thermo Fisher Scientific (1 mentions)
Calu 3, by American Type Culture Collection (1 mentions)
Ultra tmb reagent, by Thermo Fisher Scientific (1 mentions)
Bca protein quantification kit, by Abcam (1 mentions)
Triton x 100 tx 100, by Merck Group (1 mentions)
Agarose bound wga, by Vector Laboratories (1 mentions)
N acetyl d glucosamine, by Merck Group (1 mentions)
9 protocols using microwell plate
1
Cell Culture Conditions for Multiple Cell Lines
2
Serological Detection of T. cruzi Infection
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Microwell plates (Nunc Maxisorp) were coated overnight at 4°C with 50 ng protein/well of T. cruzi lysate, 2 µg/well of recombinant proteins P2β-His and CP0-His or 2 µM of synthetic peptide in 50 µL of 0.05 M carbonate buffer pH = 9.6. Plates were washed with PBS containing 0.1% Tween-20 (PBST) and then blocked with PBST containing 2.5% non-fat dry milk (PMT) for 1 h at 37°C. After washing, 50 µL of each diluted human serum (dilution 1/200 in PMT) was loaded onto plates and incubated for 1 h at 37°C. Following washing, plates were incubated with 50 µl of peroxidase-conjugated goat anti-human IgG (dilution 1/3,000 in PMT) (Sigma, St Louis, MO, USA). Enzyme activity was revealed with TMB and, OD was read at 415 nm with an Automated Plate Reader (Molecular Devices, CA, USA). All samples were tested in duplicate. Sera from 8 non-infected individuals were also included on the plate to determine the baseline level, as the OD mean value +3 SD. Antibody level is expressed as Reactivity index which was determined as the OD mean value of each serum sample/baseline value.
3
Antibody Binding Characterization of NoV VLPs
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Binding characterization of purified antibodies to NoV VLPs was carried out by ELISA. NoV VLPs were suspended in PBS at 1 μg/mL and coated in microwell plates (Nunc) for 16 h at 4°C, and the wells were blocked with 5% skim milk and 2% goat serum in PBS-Tween. Purified antibodies were diluted serially in PBS and added to the ELISA plates. The bound antibodies were detected using alkaline phosphatase conjugated goat anti-human κ or λ chain antibodies (Southern Biotech). To compare binding between different classes of antibodies, the concentrations of antibodies were adjusted to normalize for the binding sites (Fab = 1; IgG = 2; mIgA = 2 or dIgA = 4) before use in ELISA. The genotype specificity of antibody binding was determined by direct ELISA, as described above, with the following modifications: VLPs were coated at 10 μg/mL and antibodies were used at a concentration of 20 μg/mL. Plates were developed using ultra-TMB reagent (Pierce ThermoFisher; Rockford, IL), following the manufacturer’s protocol, and optical density as read at 450 nm using a SpectraMax M5 plate reader.
4
Monoclonal Anti-CD63 and Galectin-3 Binding
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Monoclonal anti-CD63 antibody (clone TS63) was from Abcam (Cambridge, UK) and biotinylated goat anti-galectin-3 (gal-3) antibodies from R&D Systems (Minneapolis, MN, USA); 3,3′,5,5′-tetramethylbenzidine (TMB), bovine serum albumin (BSA), D-lactose, and methyl- alpha D-mannopyranoside were from Sigma (St. Louis, MO, USA). Biotinylated goat anti-mouse IgG, biotinylated plant lectins, concanavalin A (ConA), SNA (Sambucus nigra agglutinin), and the Elite Vectastain ABC kit were from Vector Laboratories (Burlingame, CA, USA). Sephadex G 200 and Sephadex DEAE A-50 were from Pharmacia AB, Uppsala, Sweden. The silver stain kit and SDS-PAGE molecular mass standards (broad range) were from Bio-Rad (Hercules, CA, USA). Nitrocellulose membrane and Pierce ECL Western Blotting Substrate were from Thermo Scientific (Rockford, IL, USA). BCA Protein Quantification Kit was from Abcam (Cambridge, UK). Microwell plates were from Thermo Scientific (Roskilde, Denmark). Lymphocyte separation medium LSM-B was from Capricorn Scientific GmbH (Ebsdorfergrund, Germany).
5
Monoclonal Antibody Detection Protocol
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Monoclonal anti-CD63 antibody (clone TS63) was from Abcam (Cambridge, UK), monoclonal anti-CD81 (clone M38) and monoclonal anti-CD9 (clone MEM-61) were from Invitrogen by Thermo Fisher Scientific (Carlsbad, CA, USA), and biotinylated goat anti-galectin-3 (gal-3) antibodies were from R&D Systems (Minneapolis, USA). 3,3′,5,5′-tetramethylbenzidine (TMB), bovine serum albumin (BSA), and Triton X-100 (TX-100) were from Sigma (St. Louis, MO, USA). Biotinylated goat anti-mouse IgG, biotinylated plant lectins: Con A (Concanavalin A), wheat germ agglutinin (WGA), and the Elite Vectastain ABC kit were from Vector Laboratories (Burlingame, CA, USA). Sephadex G-200 was from Pharmacia AB (Uppsala, Sweden). The silver stain kit and SDS-PAGE molecular mass standards (broad range) were from Bio-Rad (Hercules, CA, USA). Nitrocellulose membrane and Pierce ECL Western Blotting Substrate were from Thermo Scientific (Rockford, IL, USA). Microwell plates were from Thermo Scientific (Roskilde, Denmark).
6
Glycoprotein Characterization Protocol
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Monoclonal anti-CD63 antibody (clone TS63) was from Abcam (Cambridge, UK); 3,3′,5,5′-tetramethylbenzidine (TMB), bovine serum albumin (BSA), methyl-alpha D-mannopyranoside, and N-acetyl-D-glucosamine were from Sigma (St Louis, MO, USA). Biotinylated goat anti-mouse IgG, biotinylated plant lectins: Con A, WGA, the Elite Vectastain ABC kit, and Agarose-bound WGA were from Vector Laboratories (Burlinghame, CA, USA). Sephadex G 200, CNBr-activated Sepharose 4B, and Con A were from Pharmacia AB (Uppsala, Sweden). The silver stain kit and SDS-PAGE molecular mass standards (broad range) were from Bio-Rad (Hercules, CA, USA). Nitrocellulose membrane and Pierce ECL Western Blotting Substrate were from Thermo Scientific (Rockford, IL, USA). Microwell plates were from Thermo Scientific (Roskilde, Denmark).
7
PBMC Stimulation and Cytokine Analysis
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PBMCs were obtained by gradient centrifugation of heparinised venous blood over Ficoll (Apotheek AZL, Leiden, the Netherlands) in the field laboratory in Nangapanda, Indonesia. A part of the PBMCs was fixed for flow cytometric analysis and a part was stimulated for analysis of cytokine responses. 0.4 × 106 cells were stimulated in Nunc 96-well round bottom MicroWell plates with Nunclon delta surface (ThermoFisher Scientific, Waltham, MA, USA) in 200 µL 10% foetal bovine serum (FBS; Greiner Bio-One Frickenhausen, Germany)/RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) for 96 hours with 1 × 106 PfRBCs, uninfected RBCs (uRBCs), or a medium control, after which supernatants were obtained.
8
Sunitinib Cytotoxicity in Osteosarcoma
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Osteosarcoma cells were treated with a range of concentrations of sunitinib (0, 10, 20, and 30 μM). According to operational guidelines, we used the Cell Counting Kit-8 (CCK-8) assay (MedChemExpress; NJ, the United States) to evaluate cell viability. Briefly, the second-generation osteosarcoma cells were grown on 96 well plates (5 × 103 cells/well) and exposed to different concentrations of sunitinib. Osteosarcoma cells were incubated in DMEM at 37°C with 5% CO2 for 24 h. Next, we rinsed cells with PBS and added 10 μL CCK8 to the medium for another 2 h. Finally, a microwell plate (Thermo Fisher) reader detected the absorbance at 450 nm.
9
Notch Signaling Activation Assay
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To assay ligand-induced activation of Notch target genes, each well of a 96-well MicroWell plate (Thermo Scientific, [442404]) was coated with 500 ng of DLL4-Fc or JAG1-Fc in PBS and incubated for 16–18 hr at 4°C. Lung ECs (1 × 105) from wild-type or Eogt−/− mice were plated into each well in 200 μl complete HuMedia-EG2. After 16 hr incubation at 37°C in a 5% CO2 incubator, total RNA was isolated from the stimulated ECs using TRIzol Reagent (Ambion, [15596018]) for qRT-PCR analysis.
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