MTT assay was conducted to detect cell viability. Cells were seeded into 96-well plates at a density of 2.0×103 per well and incubated in DMEM containing 10% FBS for 3–5 days. Then, 20 μL MTT were added and cells were incubated for another 4 h at 37°C, the formazan crystals were dissolved by 150 µL dimethyl sulfoxide (DMSO; Sigma). Finally, the cell viability was measured at 490 nm using Multiskan Ascent 354 microplate reader (Thermo Labsystems, Waltham, MA, USA).
Multiskan ascent 354
Manufactured by Thermo Fisher Scientific
Sourced in United States, Finland
The Multiskan Ascent 354 is a microplate reader designed for absorbance measurements. It is capable of reading 96-well and 384-well microplates. The device provides accurate and reproducible results across a wide range of applications.
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Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Kanamycin a, by Merck Group (1 mentions)
Sterile breathable film, by Corning (1 mentions)
Dimethyl sulfoxide (dmso), by Greiner (1 mentions)
Fviia, by Novo Nordisk (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Anti goat igg peroxidase, by Merck Group (1 mentions)
11 protocols using multiskan ascent 354
1
MTT Assay for Cell Viability
2
Antibiotics Sensitivity Assay
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Growth inhibition was measured as previously described [15 (link),23 (link)]. Briefly, overnight cultures of MJF376, MJF451, MJF455, and MJF612 strains carrying constructs were diluted at 1:50 (v:v) in CphM and grown, until an OD600 of 0.2 was reached. Expression was then induced by the addition of 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG) for 30 min, 10 mM stocks of compound 262 solubilized in sterile dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA) were diluted two times to achieve its final concentration in pre-warmed CphM with a final DMSO concentration of 2%, and 100 µL were added to wells of a pre-warmed, sterile 96-well flat bottom plate (Greiner bio-one, Monroe, NC, USA). Cultures were then diluted at 1:200 (v:v) in pre-warmed CphM, 100 µg/mL ampicillin, and 2 mM IPTG with diluted experimental antibiotics and were indicated, at 2× their concentration or mock (DMSO only). One hundred microliters of culture mixture with kanamycin A at a final concentrations of 0.2 µM (Sigma-Aldrich, St. Louis, MO, USA) were added to 96-well plates for a total of 200 µL, sealed with a sterile breathable film (Axygen, Union City, CA, USA), wrapped in aluminum foil and placed in a 37 °C shaker and rotated at 110 Cycles per minute for 16–17 h. OD620 was then taken with a Multiskan Ascent 354 (Thermo Fisher Scientific, Waltham, MA, USA) plate reader.
3
Cellular Proliferation Assay with C2-Ceramide
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The cellular proliferation was assessed using a WST-1 assay. Briefly, a total of 1 × 103/well and culture cells in 96-well plates in a final volume of 100 µL/well medium were treated with the indicated concentrations of C2-ceramide for 24 h respectively. BPIQ, a synthetic analog of camptothecin as a positive control [51 (link)], was used. Ten μL/well WST-1 reagent kit (Takara Biochem., Tokyo, Japan) was added and incubated for 30 min. The absorbance of the samples against a background control as blank was measured by a microplate reader (Multiskan Ascent 354, Thermo Fisher Scientific, Rockford, IL, USA) at 450 nm.
4
Quantifying Tissue Factor-Driven Coagulation
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TF-dependent procoagulant activity was measured on attached cells, supernatant or isolated EVs. Cells and pelleted EVs were kept in HBS with 1.5 mM CaCl2. The assay was started upon addition of 1 nM FVIIa (Novo Nordisk, Alphen aan den Rijn, The Netherlands) and 50 nM FX (Stago, Leiden, The Netherlands). The reaction was quenched after 30 min at 37 °C, followed by measurement of the generated amount of FXa using the chromogenic substrate Spectrozyme® FXa (Werfen, Breda, The Netherlands) and a kinetic recording spectrophotometer (Multiskan Ascent 354, Thermo Fisher Scientific). Measured rates were corrected for the number of cells present per sample. TF-blocking antibody mAb TF-5G9 was described previously [31 (link)]. Isotype-matched mouse IgG1 (TIB115) was used as control. Unlabeled human recombinant Annexin V was purchased from BioVision, Inc. (ITK diagnostics, Uithoorn, The Netherlands).
5
DPPH Radical Scavenging Assay for trans-FA
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The anti-oxidant activities of trans-FA were measured based on the scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) (#D9132, Sigma-Aldrich) free radical [34 (link), 35 (link)]. Briefly, vitamin C standards and various trans-FA concentrations were freshly prepared and diluted in methanol. Methanol (as a blank control; 10 µM) or trans-FA solution was added to 90 µL DPPH solution to yield a final trans-FA concentration of 0.15 mg/mL in a 96-well microplate. The mixture was incubated at 25 °C and protected from light. After incubation, solution absorbance was measured at 492 nm using a Multiskan Ascent 354 microplate reader (Thermo Fisher Scientific, Rockford, IL, USA). DPPH radical scavenging activity was calculated as follows: where A0 and A1 are the absorbances of the control and trans-FA solutions, respectively. Each experiment was repeated three times and found to be reproducible within experimental error margins.
6
Measuring Chondrocyte Proliferation via MTT Assay
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Cell proliferation was measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-razolium bromide (MTT) assay. Chondrocytes were seeded in 96-well plates at a density of 5×103 cells/well containing 100 µl of culture medium. The cells were transfected with 100 nM miR-34a mimic, miR-34a inhibitor or non-specific NC for 6 h. After transfection, 20 µl of MTT (5 mg/ml) (Sigma-Aldrich, St. Louis, MO, USA) was added every 24 h, followed by incubation for another 4 h at 37°C. The medium was removed, and 100 µl of dimethyl sulfoxide was added to each well to dissolve the formazan. The optical density (OD) was evaluated by measuring the absorbance at the wavelength of 490 nm, with a reference wavelength of 630 nm, using a microplate reader (Multiskan Ascent 354; Thermo Labsystems, Vantaa, Finland).
7
Optimized Immunoglobulin Quantification in Mucus
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Optimal test conditions were established based on the results obtained from two pools of positive and negative mucus samples. The final concentrations of somatic antigen used for the determination of specific immunoglobulin G (IgG) or IgA levels in mucus were 3.0 µg/ml or 5.0 µg/ml, respectively. Samples were diluted (1:100 for IgG or 1:25 for IgA) in PBS (0.8% w/v NaCl, 0.02% w/v KCl, 0.144% w/v Na2HPO4, 0.024% w/v KH2PO4; pH 7.2), and conjugate (anti-goat IgG-peroxidase [Sigma-Aldrich Inc., USA] or anti-goat IgA-peroxidase [Acris GmbH, Germany]) was diluted in PBS and used at a 1:1000 or 1:5000 dilution, respectively.
A citric acid-phosphate buffer containing 0.04% (w/v) o-phenylenediamine dihydrochloride (OPD) and 0.1% (v/v) H2O2 was used as substrate. All samples were analyzed in duplicate; the optical density (OD) was determined at a wavelength of 492 nm (Multiskan Ascent 354, Thermo Labsystems, USA) [22 (link)].
A citric acid-phosphate buffer containing 0.04% (w/v) o-phenylenediamine dihydrochloride (OPD) and 0.1% (v/v) H2O2 was used as substrate. All samples were analyzed in duplicate; the optical density (OD) was determined at a wavelength of 492 nm (Multiskan Ascent 354, Thermo Labsystems, USA) [22 (link)].
8
Glycan Analysis of CD52-Fc Isoforms
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We have previously (4 (link)) used Maackia amurensis and Sambucus nigra lectins to distinguish CD52-Fc glycans containing, respectively, sialic acid in α-2,3 and α-2,6 linkage with galactose (8 (link), 9 (link)). Here we used Maackia amurensis (MAA-I/MAL-I; Vector Laboratories, Burlingame, USA) to identify the α-2,3 linkage. A 96-well flat-bottom plate was coated with 20 μg/mL of MAL-1 overnight at 4°C and subsequently blocked with 200 μl of 1 % BSA for 1 h. After washing with PBS, CD52-Fc I, CD52-Fc II, or CD52-Fc III (20 μg/mL) were added and incubated at RT for 1 h and washed twice with PBS. After washing with PBS, 50 μl of a 1:1,000 dilution of HRP-conjugated antibody to CD52 (Campath H1; 1 μg/mL) was added and incubated at RT for 1 h. 50 μl of 3,3′5,5′-tetramethylbenzidine (TMB) substrate was added and color development stopped by addition of 50 μl of 0.5 M H2SO4. Absorbance was measured at 450 nm in a Multiskan Ascent 354 microplate photometer (Thermo Labsystems, San Francisco, USA).
9
Gametocyte Inhibition Assay for P. falciparum
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Gametocytes were induced from asexual P. falciparum NF54 parasites as described before61 (link). The parasite lactate dehydrogenase (pLDH) assay was performed as previously described59 (link),62 (link). Early and late stage P. falciparum gametocyte cultures (2% haematocrit, 5% gametocytaemia) were treated with compounds at 1 and 5 μM (primary dual-point primary screening), with methylene blue (5 µM) as positive control for inhibition. Gametocytes were treated under drug pressure for 72 h at 37 °C, followed by drug washout with culture medium and remaining pLDH activity was determined 72 h later by addition of Malstat reagent and absorbance measured with a Multiskan Ascent 354 multiplate scanner (Thermo Labsystems, Finland) at 620 nm. The percentage of viability was plotted as a function of drug concentration and curve fitting was obtained by non-linear regression analysis using a four-parameter logistic method. A full dose-response evaluation was performed for hit compounds against early and late stage P. falciparum gametocytes for three independent biological replicates. Assay performances were evaluated with average %CV at 4.54 and Z-factors at >0.5.
10
Cytotoxicity Evaluation of Chitosan Nanoparticles
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The cytotoxicity of blank chitosan nanoparticles and DOX-loaded chitosan nanoparticles was evaluated by MTT method. HepG2 cells were seeded at a density of 5 × 103 cells/well in 96-well plates and cultured overnight. The medium was then replaced with 100 μL fresh medium containing blank chitosan nanoparticles or DOX-loaded chitosan nanoparticles (Gly-CS-VE, Gly-CS-DCA, Gal-Gly-CS-VE, and Gal-Gly-CS-DCA) with different concentrations. After 48 h of incubation, the cells were washed with PBS three times, and incubated for another 4 h with fresh medium containing 20 μL MTT (5 mg/mL). Then, the medium was carefully taken away and 200 μL DMSO was added to dissolve the formazan crystals. The absorbance of each well was tested at 570 nm by a microplate reader (Multiskan Ascent 354, Thermo Fisher Scientific, Rockford, IL, USA). Cells without any sample were incubated as parallel negative controls, and the wells with added culture medium without cells served as blank controls for zero absorbance at the same time. The cell viability was calculated as the following equation:
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