The four different Bt strains were prepared at different concentrations of 0, 106, 107, 108, and 109 spores/mL. L3 instar larvae from the four different insect species (S. exigua, P. xylostella, T. molitor, and A. albopictus) were used for the Bt bioassays, in which the leaf-dipping method was used for all bioassays except for the mosquito assay. Briefly, a piece of cabbage leaf (2 cm2) was dipped into a Bt suspension for 5 min. After removing excess suspension, the treated cabbage was placed on filter paper in a Petri dish (90 × 15 mm), on which 10 randomly chosen larvae were released. After 24 h, the treated leaf was replaced with a fresh one. Data were collected daily for up to 3 days. Each concentration was replicated three times with 10 larvae per replication. For the mosquito bioassay, early L3 larvae were used for all bioassays in 1 mL of distilled water suspension containing different concentrations of BtI. For the dose–mortality test, BtI was prepared at 0, 106, 107, 108, and 109 spores/mL. Each bioassay was replicated three times. Each replication used 10 individual larvae per well of a 12-well Costar culture plate (Corning, Lowell, MA, USA). The plates were placed at 25 °C with a relative humidity of 70% in an environmental chamber. Larval mortalities were assessed 3 days after treatment.
Costar culture plate
Manufactured by Corning
Sourced in United States
The Costar culture plate is a laboratory equipment used for growing and maintaining cell cultures. It provides a sterile, controlled environment for culturing cells. The plate is made of high-quality materials and is designed to support cell growth and proliferation.
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6 protocols using costar culture plate
1
Bt Biopesticide Toxicity Assay
2
3D Co-Culture of MECs and MDCs in Matrigel
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Suspensions of 2×106 MECs and 2×106 MDCs in 500 µL DMEM were added to 0.5 mL cold Matrigel basement membrane matrix (354248, BD Biosciences, USA), mixed with cooled pipettes, and then added to the wells of a 6-well Costar culture plate (3516, Corning, USA). The plates were incubated at 37 ℃ for 30 min to allow gel formation, and then DMEM/F12 medium containing penicillin/streptomycin (15140122, Pen/Strep; 10,000 U/mL; 1%; Gibco/Thermo Fisher Scientific), 2 nmol/mL triiodothyronine, 0.4 µg/mL hemisuccinate hydrocortisone, 1% insulin-transferrin-selenium, 10 ng/mL human EGF recombinant protein, and 2% fetal bovine serum were added. The medium was subsequently changed every other day. The 3D constructs were observed under an inverted microscope (Olympus, Japan) and the experiment was repeated three times.
3
Leaf-dipping method for Bacillus thuringiensis efficacy
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The leaf-dipping method was used to determine the control efficacies of the BtA+XhE mixture against different insects, including S. exigua, S. litura, S. frugiperda, M. vitrata, P. xylostella, P. brassicae, and T. molitor, but not A. albopictus. An artificial diet was used for the dipping assay against S. litura, S. frugiperda, and M. vitrata. For P. xylostella, P. brassicae, and T. molitor bioassays, cabbage was used for the dipping assays. A constant concentration of BtA (1.25 mg (=108 spores)/mL) was mixed with XhE (4 mg/mL) for 5 min. Then, the leaf or diet was removed and kept under a clean bench for drying for 10 min and then placed into a Petri dish (90 × 15 mm). After 24 h, the treated leaf or diet was replaced with a fresh one. For the mosquito bioassay, 10 larvae were added to each well of a 12-well Costar culture plate (Corning, Lowell, MA, USA). Then, BtA+XhE was added to each well at a final volume of 1 mL. Dead larvae were counted daily for up to 3 days. Each treatment was replicated three times with 10 larvae for an experimental unit.
4
Neutrophil Adhesion and Activation Assay
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Costar culture plates (Corning Inc) were coated with 100 µg/mL poly‐L‐lysine (Solarbio) according to manufacturer's instructions before freshly isolated neutrophils (1 × 107 cells/mL) were gently added. After incubation at 37°C in 5% CO2 for 0.5‐1 hours, neutrophils were stimulated with APS‐IgG (15 µg/mL), HC‐IgG (15 µg/mL) and phorbol‐12‐myristate‐13‐acetate (PMA; 50 nmol/L; Sigma‐Aldrich, St. Louis, MO) or left untreated.
5
In silico nutrient transport modeling
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Figure 1B highlights the frequency of different cell culture vessels used for 2D monolayer cell culture and expansion. The in silico nutrient transport models were created using COMSOL Multiphysics 6 (COMSOL Inc., Burlington, Massachusetts) for the most used culture vessels. Geometries were created based on Corning™ Costar™ culture plates and SARSTEDT flasks with a standard working volume of media (6‐well = 2 ml, T‐25 = 5 ml, T‐75 = 10 ml, and T‐175 = 20 ml) and an effective diffusion coefficient of 2.8 × 10−9 m2/s for oxygen, 5.67 × 10−10 m2/s for glucose, and 5.68 × 10−10 m2/s for lactate at 37°C.31 , 32 , 33 Nutrient concentrations at the cell surface were established using a transient analysis of coupled reaction–diffusion equations together with cell proliferation kinetics.
6
3D Hydrogel Wound Healing Assay
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6-well Costar® culture plates (Corning Inc., NY, USA) were layered with 2 ml of the 3D hydrogel with or without TECA. Each well was seeded with 500,000 cells in 10% FCS/DMEM and placed at 37 °C and 5% CO2 incubator for 48 h. Wounds were induced by scratching the samples with a sterile pipette tip to leave a scratch of approximately 1 mm width. Suspended cells were removed by aspiration. Digital images were captured with an inverted microscope (Nikon Eclipse Ti-U, Nikon, Japan) and QImaging Retiga 2000R CCD digital camera (QImaging, BC, Canada). The selected fields were captured every 2 min during 24 h using phase microscopy. The distance between the selected cells and wound edge was measured. Migration rate was calculated as cell migration distance/time (µm2/h). The digitized images were analyzed using Image-J software and Pro-Plus Imaging software [49] (link), [50] (link).
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