For infection, cells were seeded on six‐well plates (Greiner, Frickenhausen, Germany) until 80% confluence was reached and infected with 5 × 105 pfu of rgRSV. After 2 hour, medium was removed and cells were incubated with substances in growth medium as indicated. Images of cells were captured using a DM IRE 220 microscope (Leica, Solms, Germany). For transfection, 5 × 105 cells were seeded on six‐well plates (Greiner) and 16 hour later transfected with 10 μg poly(I:C) using 20 μg PEI in a total volume of 1 mL Opti‐MEM I (Gibco, Thermo Fisher Scientific, Waltham, MA). After 4 hour, medium was removed and cells were treated for 24 hour with substances in growth medium as indicated.
Six well plate
Manufactured by Greiner
Sourced in Germany, Austria, United States, Cameroon, United Kingdom
Six-well plates are a type of cell culture dish used in laboratories. They provide six individual wells for culturing cells or performing various experiments. The plates are typically made of durable, transparent plastic materials that allow for visual observation and analysis of the cell cultures.
Automatically generated - may contain errors
Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Corning (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
Opti mem 1, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Cytation 5, by Agilent Technologies (1 mentions)
Jsm 7600f, by JEOL (1 mentions)
Stemfit ak02n, by Ajinomoto (1 mentions)
Black 96 well plate, by Greiner (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Leupeptin, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Gm6001, by Merck Group (1 mentions)
Human igg1, by R&D Systems (1 mentions)
Phosphate buffered saline solution pbs, by Merck Group (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
45 protocols using six well plate
1
Infection and Transfection Protocols
2
STAT1 Gene Silencing in Cells
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The siRNAs were designed and synthesized by Guangzhou RiboBio (RiboBio, Guangzhou, China). The siRNAs targeting on STAT1 gene were designed and synthesized, and the most effective siRNA (STAT1) identified by qPCR was applied in further experiments. Twenty-four hours prior to transfection, cells were plated onto a six-well plate (Greiner, Germany) at 40–60% confluence. Transfection was performed with Lipofectamine 2,000 (Invitrogen) according to the manufacturer’s protocol. The medium was replaced 4–6 h after transfection with new culture medium.
3
Quantifying Planktonic and Biofilm P. aeruginosa
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Two milliliters of M9 media were dispensed into each of three wells in a six-well plate (Greiner, North Carolina) and inoculated with 100 μL of P. aeruginosa grown overnight in LB. After 8 or 16 h incubation at 30°C, supernatant was carefully collected from all wells. Planktonic cells were collected from 2 mL of media. Planktonic cells and biofilm-associated cells attached to the bottom of the plate were washed twice with PBS. Planktonic cells were resuspended in 0.5 mL PBS buffer and dispensed into a six-well plate. Biofilms and concentrated planktonic cells were imaged using a custom filter (445/45 excitation, 510/42 emission, 482 nm dichroic) using a Cytation5 multimode reader (Biotek, Vermont). All images were taken under identical conditions, and each experiment consisted of at least three biological replicates.
4
Biofilm Formation of Streptococcus and Lactobacillus
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Streptococcus mutans and Lactobacillus sp. were cocultured overnight at 37°C in BHI and MRS broth respectively followed by dilution to a concentration equivalent to McFarland 0.5. A clean sterile cover slide was added to the wells of the six‐well plate (Greiner Bio‐One, KremsmÜnster, Austria). In each well, 250 μl of the Streptococcus mutans suspension and 250 μl of one of the Lactobacillus sp. suspension were added to 1.5 ml of BHI broth (supplemented with 0.2% sucrose) and incubated anaerobically at 37°C for 24 hrs.
A monospecies culture of Streptococcus mutans biofilm was similarly prepared except that we replaced the Lactobacillus sp. culture with uncultured MRS medium. Cover slides were gently washed with phosphate‐buffered saline (PBS) once, fixed and prepared for SEM observation (JSM‐7600F, JEOL) according to a previously published protocol30 .
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Streptococcus mutans and Lactobacillus sp. were cocultured overnight at 37°C in BHI and MRS broth respectively followed by dilution to a concentration equivalent to McFarland 0.5. A clean sterile cover slide was added to the wells of the six‐well plate (Greiner Bio‐One, KremsmÜnster, Austria). In each well, 250 μl of the Streptococcus mutans suspension and 250 μl of one of the Lactobacillus sp. suspension were added to 1.5 ml of BHI broth (supplemented with 0.2% sucrose) and incubated anaerobically at 37°C for 24 hrs.
A monospecies culture of Streptococcus mutans biofilm was similarly prepared except that we replaced the Lactobacillus sp. culture with uncultured MRS medium. Cover slides were gently washed with phosphate‐buffered saline (PBS) once, fixed and prepared for SEM observation (JSM‐7600F, JEOL) according to a previously published protocol
5
Feeder-Free Culture of iPSCs
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All iPSC lines were cultured in StemFit® AK02N (Ajinomoto) and iMatrix-511 silk (Matrixome) under feeder-free conditions30 (link). 4 × 104 cells were seeded on a six-well plate (Greiner) and passaged with the EDTA method53 (link) every 4 days. The cells were routinely tested for mycoplasma contamination. All iPSC lines generated in this study were examined for their karyotypes by LSI Medience (Tokyo, Japan).
6
Quantifying Bacterial Biofilm and Planktonic Cells
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Two milliliters of M9 media were dispensed into three wells of a six-well plate (Greiner, North Carolina) and inoculated with 100 μL of PA14 dsRed/PpvdA-gfp LB overnight culture. After incubation at 30°C for 16 h, supernatant was carefully collected from each well. Planktonic cells were collected from 1 mL of media. Cells in biofilm were collected by scraping all three wells into 1 mL of PBS buffer using a cell scraper. The two cells samples were extensively washed and resuspended in 1 mL of PBS. GFP and dsRed fluorescence from 100 μL of each sample was measured in a black 96-well plate (Greiner, North Carolina). Each experiment consisted of at least three biological replicates. Statistical significance was determined using Student's t-test.
7
Insulin Secretion in MIN6 Cells
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4 * 105 MIN6 cells in 3 mL DMEM were seeded into a six-well plate (Greiner Bio-One, Kremsmünster, Austria). After attaching to the plate for 24 h, cells were exposed to 1.5 mM OA for 24 h. One milliliter of culture medium was collected and centrifuged at 2000 rpm for 5 min at 4°C. Supernatant was collected for further analysis. PBS-washed cells were lysed with 300 µL of NP-40 buffer (10% aqueous solution, Merck Millipore, Burlington, MA, USA) supplemented with 3% v/v protease inhibitor cocktail 100× (Halt, Thermo Fisher Scientific, Waltham, MA, USA) for 20 min at 4°C. After centrifugation at 12,000 rpm for 15 min at 4°C, supernatant of cell lysate was collected. Insulin content of cell lysate and culture medium was measured by direct sandwich ELISA (DRG Diagnostics, Marburg, Germany). An aliquot of lysate samples was analyzed for total protein by Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, USA) to normalize insulin results to total protein [31 (link)]. In total, 0.1–1 mg/mL BSA was used as standard.
8
Embryoid Body Differentiation on Alvetex
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Prior to use, six-well inserts containing Alvetex® Polaris polystyrene membranes (Reprocell Europe, United Kingdom) were treated using previously established protocols. Briefly, inserts were washed in 70% ethanol, before being rinsed in sterile phosphate buffered saline (PBS) twice, and placed in a six-well plate (Greiner Bio-One, Stonehouse, United Kingdom) with the appropriate medium.
After formation, PSC derived EBs were transferred individually to the six-well inserts, with around 6–8 being placed on each insert in submerged culture. For H9 EBs, Alvetex® inserts were coated in Matrigel Growth Factor Reduced Basement Membrane Matrix prior to seeding to aid attachment. EBs were then cultured for a defined timescale, before washing in PBS and fixation in 4% paraformaldehyde (PFA, Fisher Scientific) overnight at 4°C.
After formation, PSC derived EBs were transferred individually to the six-well inserts, with around 6–8 being placed on each insert in submerged culture. For H9 EBs, Alvetex® inserts were coated in Matrigel Growth Factor Reduced Basement Membrane Matrix prior to seeding to aid attachment. EBs were then cultured for a defined timescale, before washing in PBS and fixation in 4% paraformaldehyde (PFA, Fisher Scientific) overnight at 4°C.
9
Cellular Protein Expression Analysis
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Cells (2 ×106)
were seeded in 2 mL of medium in a six-well plate (Greiner) and treated
with the desired concentration of the compounds. Cells were incubated
at 37 °C for 72 h and then lysed in whole cell extract buffer
[20 mM HEPES (pH 7.9), 20% glycerol, 50 mM KCl, 1 mM EDTA, 1 mM DTT
(Sigma-Aldrich), 400 mM NaCl, 5 μg/mL leupeptin (Sigma-Aldrich),
5 mM β-glycerophosphate, 1 mM PMSF (Sigma-Aldrich), 5 μg/mL
aprotinine (Sigma-Aldrich), 10 mM NaF, and 5 mM Na3VO4]. Protein concentrations were determined by the Bradford
protein assay. Thirty micrograms of cell lysates per treatment was
fractionated on sodium dodecyl sulfate–polyacrylamide gels
and transferred to nitrocellulose membranes (Cytiva). Then, 5% BSA
in TBS-T was used for blocking, and antibodies (ac-SMC3, HDAC8) were
diluted in TBS-T. Equal loading was confirmed by probing the same
membranes with a specific antibody for human ACTIN (1:1000, sc-47778).
were seeded in 2 mL of medium in a six-well plate (Greiner) and treated
with the desired concentration of the compounds. Cells were incubated
at 37 °C for 72 h and then lysed in whole cell extract buffer
[20 mM HEPES (pH 7.9), 20% glycerol, 50 mM KCl, 1 mM EDTA, 1 mM DTT
(Sigma-Aldrich), 400 mM NaCl, 5 μg/mL leupeptin (Sigma-Aldrich),
5 mM β-glycerophosphate, 1 mM PMSF (Sigma-Aldrich), 5 μg/mL
aprotinine (Sigma-Aldrich), 10 mM NaF, and 5 mM Na3VO4]. Protein concentrations were determined by the Bradford
protein assay. Thirty micrograms of cell lysates per treatment was
fractionated on sodium dodecyl sulfate–polyacrylamide gels
and transferred to nitrocellulose membranes (Cytiva). Then, 5% BSA
in TBS-T was used for blocking, and antibodies (ac-SMC3, HDAC8) were
diluted in TBS-T. Equal loading was confirmed by probing the same
membranes with a specific antibody for human ACTIN (1:1000, sc-47778).
10
Modulation of EphA1 Shedding in HEK Cells
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HEK transfectants were seeded at a density of 0.5 × 106 cells per well in a six‐well plate (Greiner). After 24 h, cells were treated with either 2 μg/mL ephrinA1‐Fc chimera (sEphrinA1; R&D Systems) or 2 μg/mL human IgG1 (R&D Systems) as control in DMEM/10% foetal calf serum (FCS) at 37°C with 5% CO2 for up to 3 h. In some experiments, cells were pre‐incubated with 25 μM DAPT (Enzo Life Science) or 25 μM GM6001 (Merck) or equivalent volume of DMSO (Sigma) as vehicle control prior to activation. Media were collected and stored at −80°C prior to analysis of released EphA1 and P460L by enzyme‐linked immunosorbent assay (ELISA). Cells were either stained for EphA1 protein expression and analyzed by flow cytometry or lysed and EphA1 levels analyzed by western blot.
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