The supercoiled pNS1-DsRed, pNS2-DsRed, and pVPNLS-DsRed plasmids used for transfection were prepared using an Endo-free Plasmid Purification Kit (Omega, United States) according to the manufacturer’s protocol. When cells grew to a 60–70% confluent monolayer in 6-well cell culture plates (Corning costar, United States), Lipofectamine 2000 reagent (Thermo Fisher Scientific, United States) was used to mediate transfection according to the recommendations of the manufacturer. The transfected cells were examined at a wavelength of 557 nm to detect DsRed expression.
6 well cell culture plate
Manufactured by Corning
Sourced in United States
6-well cell culture plates are a laboratory equipment used for cell culture experiments. These plates provide a standardized platform with six individual wells to grow and maintain cells in a controlled environment. The plates are designed to facilitate the simultaneous cultivation and observation of multiple cell samples.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
24 well cell culture plate, by Corning (5 mentions)
I5500, by Merck Group (3 mentions)
Matrigel, by BD (3 mentions)
Opti mem, by Thermo Fisher Scientific (3 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions)
I7018, by Merck Group (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Insulin, by Merck Group (2 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
96 well cell culture plate, by Corning (2 mentions)
Sigma 300, by Zeiss (2 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Lipid peroxidation mda assay kit, by Beyotime (2 mentions)
92 protocols using 6 well cell culture plate
1
Plasmid Transfection Optimization Protocol
2
NIR-II Imaging of Apoptotic Cells
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The CH1055-GK probe was dissolved in PBS with 5% dimethyl sulfoxide (DMSO, Gino Biomedical Technology) to a concentration of 0.5 nm μL−1. For the uptake study, apoptotic cells were randomly assigned to each group and incubated with CH1055-GK (2 nM probe per 200 μL PBS per sample) in 6-well cell culture plates (Costar, Corning, NY). For the blocking group, 1 h incubation with peptide GK (200 nM) was performed before the incubation with CH1055-GK. At different incubation times (0–24 h), cell samples were washed three times with cold PBS, and then the apoptotic cells were collected using an enzyme-based cell detachment solution in the PCR tubes (250 μl). Furthermore, the PCR tubes were centrifuged for 3 min at 400 r/min. The supernatant was then removed using a NIR-II system purchased from Suzhou NIR-Optics Technologies (Suzhou, China). The binding affinity was tested by preparing different concentrations of peptide GK in PBS (1 × 10−3-10−9 M per 200 μL PBS per sample) as the blocking agents for CH1055-GK. Apoptotic cells were incubated with the blocking agent for 1 h and then incubated with CH1055-GK (2 nM probe per 200 μL PBS per sample) for 8 h. The subsequent steps were performed as described above for the cell-uptake experiment. Finally, NIR images were collected with a NIR-II system.
3
Maintaining Human Induced Pluripotent Stem Cells
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hiPSCs were gifted by Professor Zhiguo Chen (Cell Therapy Center, Beijing Institute of Geriatrics, Xuanwu Hospital Capital Medical University) (18 (link)) and were maintained in mTeSR1 medium (Stemcell Technologies, Inc.; cat. no. 85850) in 6-well cell culture plates (Costar; Corning, Inc.; cat. no. 3516) coated with 1% vol/vol human embryonic stem cell-qualified Matrigel (Corning, Inc.; cat. no. 354277) in a 37˚C incubator at 5% CO2. hiPSCs were passaged in Dissociation Solution for human embryonic stem cells/iPSCs (ReproCELL, Inc.; cat. no. RCHETP002) at a 1:3 ratio every 7 days, according to the manufacturer's protocol.
4
Lung Fibroblast IL-1β Response
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Control lung fibroblasts (5 × 105 cells) were seeded in 6-well cell culture plates (Corning Costar) on day 1 and left overnight. On day 2, the cells were serum-starved with 2 ml of fresh medium (0.1% FBS) and treated with/without 3 ng/ml IL-1β (recombinant, expressed in E. coli, Sigma-Aldrich, I9401-5UG) for 6 h before all supernatants were collected and cells were harvested for RNA extraction.
5
Isolation and Polarization of Bone Marrow-Derived Macrophages
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The mice were sacrificed by cervical dislocation following anesthesia, and the abdomen and hind legs were sterilized with 75% ethanol. An incision was made in the midline of the abdomen, and clipped outward to expose the hind legs. Scissors were then used to remove all muscle tissue from the bones and the bones were cut at both ends to free them. The bones were crushed in a mortar with 5 ml DMEM. The femur and tibia were separated by cutting at the knee joint. BMDMs were isolated from BALB/c mice by flushing the femurs with Dulbecco's modified Eagle's medium (DMEM) (Gibco, Eggenstein, Germany). The cells were collected in 15-ml tubes and centrifuged for 10 min at 100 × g. The supernatant was removed, and the pellet was suspended in DMEM containing 20% fetal bovine serum (FBS) and 20% L929 supernatant. The cells were then seeded at 1×106 cells/well to 6-well cell-culture plates (Corning Costar, Corning, NY, USA) at 37°C in 5% CO2. After 7 days in culture, the medium was removed and the cells were cultured in RPMI-1640 medium supplemented with 10% FBS for an additional 24 h. Macrophage polarization was obtained by adding 100 ng/ml LPS plus 20 ng/ml IFN-γ (for M1 polarization), or 20 ng/ml IL-4 (for M2 polarization) in DMEM containing 10% FBS and incubating for 48 h.
6
Preadipocyte Differentiation Protocol
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Primary preadipocytes were seeded in 6-well cell culture plates (Corning Costar Corp., Cambridge, MA, USA) and cultured in BCM. After cells were ~70% confluent, the BCM was replaced by freshly-prepared differentiation culture medium 1 (DCM1) adding 0.5 mM 3-Isobutyl-1-methylxanthin (IBMX; I-7018; Sigma-Aldrich), 1 μM dexamethasone (D-4902; Sigma-Aldrich) and 1 μg/mL insulin (I-5500; Sigma-Aldrich) in BCM to induce preadipocytes differentiation. After 2 days, DCM1 was replaced with differentiation culture medium 2 (DCM2), which contained a final concentration of 1 μg/mL insulin in BCM, to maintain the differentiation state. Fresh DCM2 was replaced every other day for about 10 days until visible lipid droplets appeared in the cell, indicating that cells had completed differentiation. After differentiation, the number of mature adipocytes was 4.0 × 105 per 6-well plate.
7
HSV-2 Infection and Antibody Neutralization
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HaCat cells were infected with HSV-2(333)ZAG (0.1 PFU/cell) in 6-well cell culture plates (Costar-Corning, Kennebunk, ME, USA) for 1 h and then overlaid with media for 9 h. Cells were washed with PBS, and then heat-inactivated immune sera (1:50 dilution in incomplete DMEM) was added to cells. Plates were kept on ice for 30 min before adding 20% v/v rabbit complement. After a 4 h incubation at 37 °C in 5% CO2, cells were detached with 500 µL/well Accutase (Thermo Fisher Scientific, Waltham, MA, USA), washed twice with PBS and stained with Zombie-NIR (BioLegend, San Diego, CA, USA) for 20 min at RT. Stained cells were then fixed with 2% paraformaldehyde and read with a Cytek Aurora 5 Laser System Flow cytometer (Cytek Bioscience, Freemont, CA, USA). Data were analyzed with FlowJo Software, version 10 (FlowJo-BD, Franklin Lakes, NJ, USA).
8
Isolation and Infection of Avian Primary Cells
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Primary cell cultures were isolated from lungs of 4-week-old broiler chickens and 4-week-old Pekin ducks as previously described [20 (link)]. Cells were grown in collagen coated cell culture flasks (Costar, Corning, UK) in Dulbecco’s Modified Eagle’s Medium (DMEM) and Ham’s F12 (1:1) supplemented with 2% chicken embryo extract (Biosera, Uckfield, UK), 5% fetal bovine serum, 1% insulin-transferrin selenium (Life Technologies, Paisley, UK) and antibiotics. Monolayers of primary cells in 6 well cell culture plates (Costar) were infected with LPAI or HPAI viruses at multiplicity of infection (MOI) of 1.0. Three wells of avian cells were used for each virus infection. Mock infections were performed without virus in triplicate wells for each cell type. Cells were rinsed with phosphate buffered saline (PBS) and infected with appropriate amount of the virus in serum free infection medium comprising 2% Ultroser G (Pal Biosepra, Cedex, France), 500 ng/mL TPCK trypsin (Sigma-Aldrich, Dorset, UK) and antibiotics in Ham’s F12 medium. After 2 h incubation with the virus, the cells were washed three times with PBS and fresh medium was added.
9
Caco-2 Conditioned Medium Modulates THP-1 Cytokines
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Human CRC Caco-2 cells were seeded at a density of 3 × 10 5 cells/cm 2 onto 6-well cell culture plates (Costar, Corning, NY, USA) for 1 week at 37°C in a humidified atmosphere with 5% CO 2 and 95% air. For the collection of colorectal cancer-derived conditioned medium (CM), Caco-2 cells were grown to confluency and were washed thoroughly for one time with glucose-free standard DMEM before culturing for 8 h in standard media containing 0 or 25 mM glucose. The standard DMEM used in the study was pyruvate-and glucose-free. The Caco-2-derived CM was centrifuged to remove cell debris and stored in aliquots at -20°C until use. The Caco-2derived CM was added to PMA-differentiated THP-1 cell cultures prior to LPS challenge for 24 h. The cytokine production by THP-1 cells was determined as described below.
10
Dermal Equivalent Generation and Analysis
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Dermal equivalent was generated with a type I-A collagen mixture (Nitta Gelatin
Inc, Osaka, Japan), 8 volumes of collagen solution, 1 volume of 10×
reconstitution solution (0.022 g/mL NaHCO3, 0.0477 g/mL HEPES, 0.05 N
NaOH), and 1 volume 10× DMEM/F12 (3:1) with IGF (1.5 × 105 cells).
Collagen mixture was loaded onto filter inserts (3 µm pore size, 12 mm in
diameter; Millipore) and placed into 6-well cell culture plates (Costar). The
collagen mixture was solidified by 24 hours incubation at 37°C; cells (1 ×
106 cells) were then loaded onto the collagen mixture. Complete
medium in the presence or absence of EEDC was added to the chamber. During the
experiment, an air-liquid interface microenvironment was generated by removing
excess media from the top of the cell layer. Two weeks later, the dermal
equivalent was removed from the filter insert and transferred to formalin
solution and histologically examined by hematoxylin and eosin staining.
Inc, Osaka, Japan), 8 volumes of collagen solution, 1 volume of 10×
reconstitution solution (0.022 g/mL NaHCO3, 0.0477 g/mL HEPES, 0.05 N
NaOH), and 1 volume 10× DMEM/F12 (3:1) with IGF (1.5 × 105 cells).
Collagen mixture was loaded onto filter inserts (3 µm pore size, 12 mm in
diameter; Millipore) and placed into 6-well cell culture plates (Costar). The
collagen mixture was solidified by 24 hours incubation at 37°C; cells (1 ×
106 cells) were then loaded onto the collagen mixture. Complete
medium in the presence or absence of EEDC was added to the chamber. During the
experiment, an air-liquid interface microenvironment was generated by removing
excess media from the top of the cell layer. Two weeks later, the dermal
equivalent was removed from the filter insert and transferred to formalin
solution and histologically examined by hematoxylin and eosin staining.
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