MDCK cells (100 µL/well) were seeded at a concentration of 2 × 105 cells/mL in 96-well cell culture plates (Sigma) and incubated at 37 °C for 12 h. The mAbs were diluted to a starting concentration of 30 µg/mL in PBS and serially diluted 1:2 in UltraMDCK media (Lonza) supplemented with tosyl phenylalanyl chloromethyl ketone-treated trypsin (infection media; Sigma) at a concentration of 1 µg/mL, in 96-well cell culture plates (Sigma). The viruses were diluted to a concentration of 100 × TCID50/50 µL (A/Hunan/02285/2017 (Hunan), A/feline/New York/16-040082/2016 (New York), A/Hong Kong/2014/2017 (Hong Kong)) in infection medium. Next, 60 µL of virus dilution was incubated with 60 µL of mAb serial dilution and incubated on the shaker at room temperature for 1 h. The plates were incubated at 33 °C for 48h (New York) or 72 h (Hunan, Hong Kong). The readout was performed by the means of classical hemagglutination assay. This readout was chosen because it is more objective than assessment of cytopathic effects but easier to perform than staining for virus antigen (e.g., for nucleoprotein). In brief, chicken RBCs (Lampire Biological Laboratories) was diluted to a concentration of 0.5% in PBS and added to 50 µL of cell supernatant in v-bottom plates (Corning). After 45–60 min the plates were scanned and the results analyzed in Microsoft Excel and GraphPad Prism 7.
96 well cell culture plate
Manufactured by Merck Group
Sourced in United States
The 96-well cell culture plates are a versatile laboratory equipment used for a variety of cell-based experiments. The plates feature a grid of 96 individual wells, providing a standardized platform for culturing cells, performing assays, and screening various treatments or conditions. The plates are designed to maintain optimal cell growth and enable consistent, reproducible results.
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Lab products found in correlation
V bottom plate, by Corning (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Versamax tunable microplate reader, by Molecular Devices (2 mentions)
Trypsin, by Merck Group (1 mentions)
Ultramdck media, by Lonza (1 mentions)
Fecl3 6h2o, by Merck Group (1 mentions)
Annexin 5 fitc, by Merck Group (1 mentions)
Cell culture flask, by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Trypan blue, by Merck Group (1 mentions)
Nh4oh, by Merck Group (1 mentions)
Ferrous chloride tetrahydrate, by Merck Group (1 mentions)
Phenol red free dmem, by Merck Group (1 mentions)
Tpck treated trypsin, by Merck Group (1 mentions)
Ultramdck medium, by Lonza (1 mentions)
Infinite m200, by Tecan (1 mentions)
Cck 8 reagent, by Dojindo Laboratories (1 mentions)
8 protocols using 96 well cell culture plate
1
Influenza Virus Neutralization Assay
2
Influenza Virus Neutralization Assay
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RDE inactivated serum samples (dilution of 1:10) were serially diluted 2-fold in 1xMEM, supplemented with TPCK-treated trypsin at a concentration of 1 μg/ml, in 96-well cell culture plates (Sigma). The A/Wyoming/03/2003 virus was diluted to a concentration of 100 50% cell culture infectious doses (TCID50) in infection medium. Sixty microliters of serially diluted serum was incubated with 60 μl of virus dilution for 1 h at room temperature on a shaker. MDCK cells were washed once with 220 μl of PBS, and 100 μl of the virus-serum mixture was added to MDCK cells. The cells were incubated for 48 h at 33°C. After 48 h, the readout was performed by the means of a hemagglutination assay as described above.
3
Cell Viability and Apoptosis Assay
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VBL sulfate, DEX (15 kDa), ferric chloride hexahydrate dimethyl sulfoxide (DMSO), FeCl3·6H2O, ferrous chloride tetrahydrate (FeCl2·4H2O), the MTT agent, NH4OH (25% of ammonia), and FA were purchased from Merck (Darmstadt, Germany). Annexin V-FITC, propidium iodide (PI), trypan blue, 96-well cell culture plates, and cell culture flasks were purchased from Sigma-Aldrich (St. Louis, MO, USA). The PANC-1 and H6c7 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).
4
Cytotoxicity Evaluation of Bacterial Strains
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The cytotoxicity of the selected strains was assessed by MTT assay. The mouse pre-adipocyte 3T3-L1 cells were seeded into 96-well cell culture plates (Sigma-Aldrich, St. Louis, MO, USA) at a density of 1 × 104 cells/well and incubated at 37 °C and 5% CO2 condition. After 24 h, the medium was replaced with maintenance medium containing each of the heat-killed strains at a concentration of 108 CFU mL−1 and incubated for 24 or 48 h. The medium was subsequently removed, and the remaining cells were used for the MTT assay. A 10-μL aliquot of a 12 mM stock solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, St. Louis, MO, USA) and phenol-red free DMEM (Sigma-Aldrich, St. Louis, MO, USA) was added to each well. After incubation for 3 h at 37 °C and 5% CO2, the supernatant was discarded, followed by the addition of 50 μL of dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA) to each well for dissolution of the formed formazan crystals in viable cells. The culture plate was then incubated for 30 min in dark. The absorbance of each well was measured at 575 nm using a VersaMax™ tunable microplate reader (Molecular Device, San Jose, CA, USA).
5
Hemagglutination Assay for Influenza H7N9 Virus
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Serum samples were treated with receptor-destroying enzyme (RDE; Denka Seiken) for 18 h at 37°C. To stop RDE treatment, sodium citrate (2.5%) was added and serum was incubated at 56°C for 1 h. The inactivated serum samples (dilution of 1:10) were serially diluted 2-fold in UltraMDCK medium (Lonza), supplemented with tosyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (infection medium; Sigma) at a concentration of 1 µg/ml, in 96-well cell culture plates (Sigma). The A/Shanghai/1/2013 H7N9 virus was diluted to a concentration of 100 50% cell culture infectious doses (TCID50) in infection medium. Sixty microliters of serially diluted serum was incubated with 60 µl of virus dilution (1,250 PFU/60 µl) for 1 h at room temperature on a shaker. MDCK cells were washed once with 220 µl of PBS, and 100 µl of the virus-serum mixture was added to MDCK cells. The cells were incubated for 48 h at 33°C. The readout was performed by the means of a hemagglutination assay. In brief, chicken red blood cells (RBCs; Lampire) were washed once with PBS and diluted to a concentration of 0.5% RBCs in PBS, and 50 µl of RBCs was added to 50 µl of cell supernatant in V-bottom plates (Corning). The plates were kept at 4°C for 30 to 45 min and scanned, and the results were analyzed in Microsoft Excel and GraphPad Prism 7.
6
Cytotoxicity Assessment of H2O2 in Human Cells
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The cytotoxicity of H 2 O 2 in HT-29 and HaCaT cells was assessed using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The human HT-29 epithelial cells and human HaCaT keratinocytes were seeded in 96-well cell culture plates (Sigma-Aldrich) at a density of 1 × 10 4 cells/well and were incubated at 37°C and 5% CO 2 condition. After 24 h, both media were replaced with maintenance medium containing lipopolysaccharide (LPS) and H 2 O 2 at various concentrations and heatkilled strains (20 min, 80°C in PBS) at a concentration of 10 8 colony-forming units (CFU) mL - 1 and incubated for 2 h. The medium was subsequently removed, and the remaining cells were used for the MTT assay. A 10 µL aliquot of a 12 mM stock solution of MTT (Sigma-Aldrich) and phenol-red-free RPMI or DMEM maintenance medium were added to each well. After incubation for 2 h at 37°C and 5% CO 2 , the supernatant was discarded, followed by the addition of 100 µL of dimethyl sulfoxide (DMSO) (Sigma-Aldrich) to each well for dissolution of the formed formazan crystals in viable cells. The culture plates were incubated for 30 min in the dark. The absorbance of each well was measured at 575 nm using a VersaMax tunable microplate reader (Molecular Devices).
7
Antimicrobial Activity of 3D Scaffolds
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To test the antimicrobial activities of the 3D scaffolds, we used S. aureus and E. coli as model microorganisms. To investigate the inhibitory effect of the different 3D scaffolds, we employed a disc diffusion method, whereby we first spread 100 mL of 105 CFU S. aureus or E. coli suspension on a nutrient agar plate, and placed each disc to be tested on agar for 16 h incubation at 37 °C. Then, we pressed the different 3D scaffolds into the discs and incubated them for 24 h at 37 °C, after which we examined the plates for the presence of inhibition zones around the samples. For the different 3D scaffolds, to determine their relative bacteria killing efficiencies, we mixed the scaffolds with 5 mL of S. aureus and E. coli in an LB culture medium (4.0 × 104 bacteria per mL), respectively, and cultured them for 24 h. The bacterial growth was measured as turbidity at an optical density at OD 600 nm, using on a Microplate Reader (Infinite M200, TECAN) and a 96-well cell culture plate (Sigma). We used a bacterial suspension without 3D scaffolds as the control, and obtained all results in triplicate.
8
Measuring Cell Viability with Zhishi and APAP
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The cells were first plated to a 96-well cell culture plate at a density of 1.0 × 104 cells/well. Then, Zhishi was added to a 96-well cell culture plate (Sigma, St. Louis, MO, USA) of BRL-3A entering the logarithmic growth phase, and incubated at 37°C for 48 hours in a 5% CO 2 incubator. The cells were washed twice with PBS, and then the new medium, Zhishi and APAP, were added to a 96-well cell culture plate. The cells were incubated with Zhishi, APAP in a cell incubator at 37 ° C, 5% CO 2 for 24 hours. Next, add 10 μl of cck8 reagent (Dojindo, Tokyo, Japan) to each well, then incubate for 3 hours in a cell culture incubator. Finally, the absorbance of each well was measured by microplate reader at 450nm.
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