Pure virus was isolated using a plaque assay on double layer agar (DLA). A 150 μl volume of tenfold serially diluted virus particles (10 -4 to 10 -8 dilutions) was seeded, along with a negative control, into 6-well plates (Sigma-Aldrich) containing confluent monolayers of RD cells (1 × 10 5 cells/cm 2 ). After absorption of the viruses at 37 °C for 1 h, 3 ml of 2x DMEM (Bio-Idea, Iran) was mixed with an equal volume of 1.5% purified cell culture grade agar (Sigma-Aldrich, cat NO 1296) supplemented with 1% heat inactivated fetal bovine serum (FBS, Bio-Idea, Iran), overlaid as a monolayer in the 6-well plates (Sigma-Aldrich), and incubated overnight at 37 °C in 5% CO 2 . Another 3 ml of 2x DMEM containing 1.5% agar was overlaid onto the 6-well plate and stained with 0.01% neutral red (Sigma-Aldrich, cat. NO. 4638). The plate was sealed with aluminum foil to protect it from light and incubated overnight at 37 °C in 5% CO 2 . Three plaques were picked up and passaged three times in a 25 cm 2 flask containing confluent RD cells. Incubation was continued until CPEs were observed, and the culture was then stored at -70 °C for further study. 13
6 well plate
Manufactured by Merck Group
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6-well plates are a type of laboratory equipment used for cell culture and other biological applications. They provide a standardized platform with six individual wells to perform experiments or assays. The plates are typically made of polystyrene or other inert materials, allowing for optimal cell growth and experimentation.
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Lab products found in correlation
Inverted microscope, by Olympus (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Lipofectaminetm 2000, by Thermo Fisher Scientific (2 mentions)
Mycoplasma free, by Southern Biotech (2 mentions)
Capped t25 flasks, by Greiner (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Gemini Bio (2 mentions)
24 well plate, by Corning (2 mentions)
L glutamine, by Quality Biological (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Trypsin edta solution, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Lonza (1 mentions)
Dispase 2, by Roche (1 mentions)
Kgm cd, by Lonza (1 mentions)
Propidium iodide, by BD (1 mentions)
Facscalibur system, by BD (1 mentions)
Annexin 5 fitc, by BD (1 mentions)
Cellquest pro, by BD (1 mentions)
28 protocols using 6 well plate
1
Plaque Assay for Virus Isolation
2
Culturing I. scapularis Cell Lines
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The I. scapularis IDE12 and ISE6 cell lines were obtained from Dr. Ulrike Munderloh at University of Minnesota. Cells were cultured in L15C300 medium supplemented with 10% heat inactivated fetal bovine serum (FBS, Sigma), 10% tryptose phosphate broth (TPB, Difco), 0.1% bovine cholesterol lipoprotein concentrate (LPPC, MP Biomedicals) at 34°C in a non-CO2 incubator. ISE6 cells were grown to confluency in capped T25 flasks (Greiner bio-one) and either seeded at 3×106 cells/well in 6-well plates (Millipore Sigma) or 3×105 cells/well in 24-well plates (Corning). IDE12 cells were also grown to confluency in T25 flasks and either seeded at 1×106 cells/well in 6-well plates (Millipore Sigma) or 3×105 cells/well in 24-well plates (Corning). The human leukemia cell line, HL-60, was obtained from ATCC and maintained in RPMI-1640 medium with L-Glutamine (Quality Biological) supplemented with 10% FBS (Gemini Bio-Products) and 1% GlutaMax (Gibco), in vented T25 flasks (CytoOne) at 37°C and 5% CO2. All cell cultures were confirmed to be Mycoplasma-free (Southern Biotech).
3
Cultivation of Tick Cell Lines
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The I. scapularis IDE12 and ISE6 cell lines were obtained from Dr. Ulrike Munderloh at University of Minnesota. Cells were cultured in L15C300 medium supplemented with 10% heat inactivated fetal bovine serum (FBS, Sigma), 10% tryptose phosphate broth (TPB, Difco), 0.1% bovine cholesterol lipoprotein concentrate (LPPC, MP Biomedicals) at 34 °C in a non-CO2 incubator. ISE6 cells were grown to confluency in capped T25 flasks (Greiner bio-one) and either seeded at 3 × 106 cells/well in 6-well plates (Millipore Sigma) or 3 × 105 cells/well in 24-well plates (Corning). IDE12 cells were also grown to confluency in T25 flasks and either seeded at 1 × 106 cells/well in 6-well plates (Millipore Sigma) or 3 × 105 cells/well in 24-well plates (Corning). The human leukemia cell line, HL-60, was obtained from ATCC and maintained in RPMI-1640 medium with L-Glutamine (Quality Biological) supplemented with 10% FBS (Gemini Bio-Products) and 1% GlutaMax (Gibco), in vented T25 flasks (CytoOne) at 37 °C and 5% CO2. All cell cultures were confirmed to be Mycoplasma-free (Southern Biotech).
4
Transfection of A549 Cells with GGA3
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After the A549 cells density reached about 80%, cells were processed for transfection. Cells maintained until mid-log phase into 6-well plates (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), and fresh cell culture medium was infused two hours prior to transfection. PcDNA3.1-GGA3 group was transfected with pcDNA3.1-GGA3 (synthesized by GeneChem, Shanghai, China), and the control group was transfected with pcDNA3.1 empty vector by Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in line with the supplier’s instruction. Ten µL Lipofectamine 2000 was dissolved in 250 µL of serum-free antibiotic-free medium, mixed gently and allowed to stand for 5 minutes at room temperature. Then 5 µL plasmid was dissolved in 250 µL serum-free antibiotic-free medium. Mixed the liposome and plasmid mixture gently within 30 min. Then discarded the medium from the 6-well plate and washed the cells twice with PBS. Added 500 µL of liposome and plasmid mixture to each well of cells, gently mixed and incubated in incubator for 6 h, replaced the mixture with cell culture medium. After 24 h incubation, we can observe the expression of plasmids or proceed with follow-up experiments.
5
Isolation and Expansion of Human Keratinocytes
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Four lines of human KCs (see information in Supplementary Table S1 ) were isolated from the waste foreskin of healthy subjects that were donated with informed consent and approved by the ethics review board of SingHealth. Briefly, the collected skin tissue was washed in phosphate-buffered saline (PBS, Lonza, Walkersville, MD, USA) and incubated in 10 mL of 2.5 mgmL−1 Dispase II (Roche, Mannheim, Germany) in Dulbecco’s modified Eagle medium (DMEM, Gibco, Thermo Fisher Scientific, Grand Island, NY, USA) and left overnight at 4 °C. The following day, epidermis was mechanically separated from dermis with fine forceps and incubated in 0.05% trypsin-EDTA solution (Gibco) for 15 min at 37 °C. Upon cellular dissociation, trypsin activity was reduced by diluting the solution with three volumes of fresh DMEM. KCs were then collected through centrifugation and resuspended in KGM-CD (Lonza) for downstream culture or cryopreservation. KCs were seeded at a density of 9 × 104 cells/cm2 on 6-well plates (Corning, Sigma-Aldrich, New York, NY, USA) pre-coated with laminin 511 [27 (link)] and cultured in KGM-CD at 37 °C/7.5% CO2 for further expansion. Cells between passages 2 to 6 were used for the in vitro experiments in this study.
6
Wound Healing Assay in 6-well Plate
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5 × 105 cells were cultured in each well of 6-well plates (Sigma) in serum-free RPMI-1640 medium until 100% confluency was reached. The layer of cells were scratched with a sterile 10 μl Eppendorf tip (Sigma), washed 3 times with PBS, and then incubated in fresh serum-free RPMI-1640 medium for 24 h. An inverted microscope (Olympus) was used to examine and photograph random fields.
7
Apoptosis Analysis of BT-3-CD133+ Cells
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To further study the apoptosis of BT-3-CD133+ cells, an optimized number of cells (2 × 105 cells) were seeded into 6-well plates (Sigma-Aldrich, Saint Louis, MO, USA), and the cells were treated with PEI-MSNs for 24 h. Cells were then harvested, washed twice with PBS, and once with 1 × Binding Buffer ((5 mM HEPES, 70 mM NaCl, 2.5 mM CaCl2, pH 7.4) in 2%FCS/PBS (w/v), 0.01% NaN3). Cells were stained with Annexin V-FITC (BD Pharmingen, San Jose, CA, USA) and incubated at RT for 20 min. Cells were then washed twice with Binding buffer. Twenty seconds prior to analysis with the FACS Calibur system, propidium iodide (PI; BD Pharmingen) was added to the sample. The data were analyzed with CellQuest Pro (BD Biosciences) or FlowJo (TreeStar Inc., Ashland, OR, USA).
8
Apoptosis Detection by Flow Cytometry
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After 96 h treatment, cells grown in 6 well plates (Sigma) were collected and washed in phosphate buffered saline (PBS). Cells were then incubated with 5 μL Annexin V-APC and 7-AAD stain (BD Biosciences, Mississauga, ON) for 15 min at room temperature in the dark. Controls were prepared by adding no stains, Annexin V only and 7-AAD only. 400 μl of binding buffer (BD Biosciences) was added to the samples. Samples were immediately analyzed by flow cytometry using a BD LSR Fortessa. Viable (Annexin V-/7-AAD-) and apoptotic cells (Annexin V+/7-AAD- and Annexin V+/7-AAD+) were quantified as a percent of total cells.
9
Inhibiting p53-directed Transcription In Vivo
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Decoy oligonucleotides containing the palindromic p53 cis-element 5′-RRRCWWGYYY-3′ (R = A or G, Y = C or T, W = A or T), which allows self-hybridization and formation of a duplex hairpin that competes with p53 enhancers for binding of transcription factors, were used to inhibit p53-directed transcription in vivo, as previously described by us57 (link). The sequences of the p53 decoy and control phosphorothioate oligonucleotides (underscored) (Sigma-Aldrich) were as follows: p53 decoy, 5′-GGACA TGCC CGGGC ATGTCC-3′; control nonsense sequence, 5′-CTA GCTA GCTA GCTA GCTA GCTAG-3′. Cells were seeded at a density of 106 cells onto in 100 mm Petri dishes (Sigma-Aldrich), differentiated as above. After that cells were collected and 800 μl of 2.5 × 107 cell/ml, electroporated in presence of 500 μg sheared salmon sperm DNA (Sigma-Aldrich) and 1 μM of each phosphorothioate oligonucleotide at 330 V and 1000 uF using an Electroporator II (Invitrogen). Next, cells were plated onto 6-well plates (Sigma-Aldrich), grown in a complete medium overnight followed by its replacement with 1% FCIII containing 100 μM OT for 72 h and analysis using RT-PCR as said above.
10
Gadd3-targeting siRNA and Tunicamycin Treatment in HeLa Cells
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