DF-1 cells were seeded into each well of 6-well plate (Costar 3516, Corning, NY) at 1 × 104 cells at 24 hrs prior to virus inoculation. A total of six individual wells were prepared for each sampling time point of both mock- and caIBDV- infected groups. The purified caIBDV was diluted to 10 multiplicity of infection (m.o.i.) per ml with DMEM-HG without serum supplement. Before inoculation, culture medium was discarded and the cells were rinsed with 1 × phospate-buffered saline once. One ml of diluted caIBDV was applied into each well of the virus-infected group, whereas DMEM-HG without serum supplement was added in the mock-infected group. At 0, 6, 12 hrs post-infection (hpi), medium was aspirated from the wells of mock- and caIBDV-infected groups. One ml of SV RNA Lysis Buffer (Promega, WI) was added into each well and the cells were dispersed and lysed with repeated pipetting. A total of six individual cell lysates were collected from each treatment group at designated time points and kept at -80°C for total RNA extraction thereafter.
Costar 3516
Manufactured by Corning
Sourced in United States
The Costar 3516 is a 96-well microplate designed for cell culture and cell-based assays. It features a clear polystyrene construction with a flat bottom to provide optimal optical properties for various analytical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Sv rna lysis buffer, by Promega (1 mentions)
Rna lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Bca kit, by Merck Group (1 mentions)
Dznep, by Cayman Chemical (1 mentions)
Neurobasal a, by Thermo Fisher Scientific (1 mentions)
Picm03050, by Merck Group (1 mentions)
Ly294002, by Selleck Chemicals (1 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
Penicillin and streptomycin, by Corning (1 mentions)
M6494, by Thermo Fisher Scientific (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Corning (1 mentions)
Trypsin edta solution, by Corning (1 mentions)
Cis diammineplatinum 2 dichloride cisplatin, by Merck Group (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
11 protocols using costar 3516
1
Quantifying IBDV Viral Infection in DF-1 Cells
2
Knocking Down SENP1 and CDX2 in LNCaP Cells
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LNCaP cells were seeded at a density of 2.0 × 105 cells/well in a 6- or 96-well culture plate (COSTAR#3516; Corning, Inc., Corning, NY) and transfected when cell confluency reached 70%. RNA Lipofectamine 2000 (Invitrogen) was used to transfect the cells with small interfering RNAs (siRNAs) to SENP1 or CDX2 according to the manufacturer's instructions. Discarded the transfection after 6 h.Then, the cells were washed with serum-free DMEM, and cultured in DMEM supplemented with 10% FBS. At 48 h after transfection, the cells were harvested for further studies. The siRNAs were designed and synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). SiRNA for SENP1 sense: 5′-GAAACAGCCGAAGCCUUUAdTdT-3′; anti-sense: 5′-UAAAGACUUCGGCUGUUUCdTdT-3′; and siRNA for CDX2: sense 5′-GAAGAAGTTGCAGCAGCAA-3′; anti-sense: 5′-UUGCUGCUGCAACUUCUUC-3′.
3
Screening H. pylori Biofilm-Defective Mutants
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A Tn-7-based H. pylori G27 transposon mutant library43 (link) was grown overnight in BB10 supplemented with 25 μg of chloramphenicol, diluted to an OD600 of 0.15 and then used to fill duplicate wells of a 6-well plate (Costar® 3516, Corning, Corning, NY, USA). After an hour of incubation, media containing non-attached, planktonic bacteria and/or potential biofilm-defective mutants was removed, and transferred to a new sterile 6-well plate and incubated again. The procedure was repeated at different incubation time (i.e., 2, 3, 24, 48, or 72 h). After 72 h, this enriched biofilm-defective sample was plated on CHBA media and individual colonies were isolated and stored at −80 °C. A total of 97 potential biofilm-defective mutants was isolated from this enrichment approach, and subsequently tested for abnormal biofilm formation using the crystal violet biofilm assay described above.
4
Bacterial Adhesion Assay with UDMA
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For bacterial adhesion assays, cells grown in the presence and absence of UDMA were washed with TBS and then resuspended in TBS to an OD600 of 0.5. The cell suspensions were then labeled with 10 μM SYTO 13 for 20 min. After that, a polystyrene microplate (Costar 3516, Corning Inc., Corning, NY, USA) was prepared with saliva, as described above, and 150 μL of the labeled cells was added into each well and incubated for 3 h. The wells were subsequently rinsed with TBS, and the optical density was measured using a microplate reader (excitation: 485 nm; emission: 528 nm).
5
Extracellular Matrix Analysis of Dual-Species Biofilms
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For this assay, dual-species biofilms were grown in six-well plates (Costar® #3516, Corning Inc., Corning, NY, USA) containing 4 mL of the microbial suspension, as previously detailed. After the last treatment, the biofilms were resuspended in 0.85% NaCl, scraped from the wells, and the liquid phase of the extracellular matrix was extracted by sonication (for 30 s at 30 W) [37 (link)]. Protein determination of the extracellular matrix was performed by the bicinchoninic acid method (Kit BCA; Sigma-Aldrich), using bovine serum albumin as the standard [37 (link)]. The carbohydrate content was quantified, as detailed by Dubois et al. [38 (link)], with glucose as the standard. For the evaluation of the nucleic acid content, 1.5 mL of the liquid phase of the extracellular matrix was spectrophotometrically analyzed (at 260 and 280 nm) in a Nanodrop Spectrophotometer (EON Spectrophotometer of EON, Biotek, Winooski, VT, USA) [39 (link)]. Protein, carbohydrate, and nucleic acid values were expressed as mg/g dry weight of biofilm.
6
Retinal Explant Culture and Treatments
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Retinal explants obtained from newborn rd1 and wt mice were cultured using a protocol from previously described methods10 (link), 19 (link), 62 (link). Briefly, both eyes were enucleated and retinas were carefully peeled away from the retinal pigment epithelium, and radial cuts were made to flatten the retina. The flattened retina was transferred to the membrane of a Millicell insert (Millipore, PICM03050) with the photoreceptors facing down. The insert was placed into the wells of a 6-well plate (Costar 3516, Corning), each contained 1300 μl of retinal explant media, which was replaced every 2 days, and was maintained in a 37 °C incubator with 5% CO2 for 10 days. The retinal explant basal medium was serum-free and made from Neurobasal A, DMEM/F12 medium, and N2/B27 supplements (Life Technologies, USA). Cultured explants were either exposed to DZNep (Cayman Chemical, 13828, Ann Arbor, USA) treatment (0.1 μM, 0.5 μM, 1 μM, 1.5 μM), LY294002 (Selleckchem, S1105, Houston, TX, USA) treatment (20 μM), or kept as PBS-treated controls.
7
SH-SY5Y Dopaminergic Neuron Culture
8
Cisplatin Exposure Effects on Zebrafish Larvae
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Cisplatin (cis-Diammineplatinum (II) dichloride) was purchased from Sigma-Aldrich, St. Louis, MO, USA, and was prepared following the manufacturer’s instructions. Cisplatin working solutions were prepared in E3 medium at final concentrations of 0.025, 0.05, 0.10, 0.15, and 0.25 mg/mL for the experiments. Cisplatin was added to the embryonic medium of the larvae at 7 days post-fertilization (dpf) and was maintained until the end of the experiment. The larvae were placed in 6-well plates (Costar 3516, Corning Incorporated, New York, NY, USA) with 20 larvae per well. The pH of the medium was measured to verify that there was no variation after adding the drug, and it remained between 7.2 and 7.3. The larvae survival was monitored daily from 7 dpf until 10 dpf. Following the LD50 determination, further analyses were conducted using a cisplatin concentration of 0.10 mg/mL with an exposure duration of 48 h.
9
PRMT5 and TXNIP Knockdown Protocol
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siRNAs targeting different sequences of PRMT5 and TXNIP were used for knockdown experiments (GenePharma, Shanghai, China). Cells were cultured at a density of 2 × 105 cells/well in a 6-well plate (Costar 3516, Corning, NY, USA) overnight. siRNAs targeting PRMT5 and TXNIP were transfected into cells using Lipofectamine 2000 (Invitrogen, CA, USA) for 4 h. Cells were then cultured in fresh complete medium for 3 days. The targeting sequences of the above siRNAs are specified in the supplementary information (Supplementary Table 3 ).
10
Bacterial Infection Assay in Cell Culture
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The cell culture plates (6 well tissue culture plate, Costar 3516, Corning, NY, USA) were washed twice with DMEM and then 125 microlitre of bacterial suspension was added along with DMEM in each well in duplicates.
One positive well was kept to check for contamination containing only media.
The plates were incubated at 37 ℃ for 2 hours in 5%CO2 incubator. After incubation, the cells were washed twice with DMEM and then 1 millilitre of DMEM with 50 microgram/millilitre gentamicin was added to each well to prevent contamination of wells.
The plate was incubated at 37℃ in 5% CO2 incubator for 30 mins and then washed twice with DMEM. Wells were then overlaid with DMEM growth medium (1.2%) [1millilitre per well] and incubated for 3 days at 37℃ in 5% CO2 incubator.
One positive well was kept to check for contamination containing only media.
The plates were incubated at 37 ℃ for 2 hours in 5%CO2 incubator. After incubation, the cells were washed twice with DMEM and then 1 millilitre of DMEM with 50 microgram/millilitre gentamicin was added to each well to prevent contamination of wells.
The plate was incubated at 37℃ in 5% CO2 incubator for 30 mins and then washed twice with DMEM. Wells were then overlaid with DMEM growth medium (1.2%) [1millilitre per well] and incubated for 3 days at 37℃ in 5% CO2 incubator.
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