bEnd.3 brain endothelial cells (ATCC, CRL-2299) were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. Cells were adhered in monolayers and, when confluent, were harvested using trypsin-EDTA and 3500 cells/well were then seeded in PET membrane filters (1 μm pore size) and pre-coated with fibronectin bovine plasma (3 μg/mL) for 24-well cell culture plates (BD Falcon). Cells were allowed to reach a confluent monolayer for 10 days and medium changed every two days, which allowed the formation of stable tight junctions mimicking the natural BBB [50 (link)].
24 well cell culture plate
Manufactured by BD
Sourced in United States, Switzerland
The 24-well cell culture plates are a versatile laboratory equipment designed for the growth and maintenance of cells. These plates provide a standardized format with 24 individual wells, allowing for parallel experiments or the cultivation of multiple cell lines simultaneously. The plates are made from high-quality, cell culture-treated polystyrene material, ensuring optimal cell attachment and growth conditions. The core function of these plates is to provide a controlled and sterile environment for the culturing of cells, enabling researchers to conduct various cell-based experiments and analyses.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Nr8383, by LGC (2 mentions)
Tpp cell scraper, by Techno Plastic Products (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Calcein, by Corning (1 mentions)
Matrigel, by Corning (1 mentions)
Ebm 2, by Lonza (1 mentions)
Egm 2, by Lonza (1 mentions)
25 cm2 cell culture flask, by Corning (1 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
Nutrient mixture f 12 dmem f12, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Corning (1 mentions)
Its premix, by Corning (1 mentions)
Modulator incubator chamber, by Embrient Inc (1 mentions)
Culture flasks, by BD (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Bm hmscs, by Lonza (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
17 protocols using 24 well cell culture plate
1
Establishing bEnd.3 Brain Endothelial Cell BBB
2
Macrophage Cytokine Release Assay
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The human leukemic cell line THP-1 [31 (link)] (ATCC, TIB-202) was used to model macrophage function. The cells were cultured in RPMI-1640 supplemented with 10% FBS and 1% P/S at 37 °C, 5% CO2 humidified air.
For the cytokine release experiments, the cells were seeded in 24-well cell culture plates (Falcon; 0.5 million cells per well) and differentiated into macrophage-like cells using 100 ng/mL PMA for 48 h. After the differentiation period, the cells were treated with various concentrations of the test substances (coffee charcoal extract: 1–500 µg/mL; pure substances: 0.01–100 µM). Budesonide (1 nM) served as a positive control for anti-inflammatory activity. Concomitantly, 100 ng/mL LPS was applied as inflammatory stimulus.
For the cytokine release experiments, the cells were seeded in 24-well cell culture plates (Falcon; 0.5 million cells per well) and differentiated into macrophage-like cells using 100 ng/mL PMA for 48 h. After the differentiation period, the cells were treated with various concentrations of the test substances (coffee charcoal extract: 1–500 µg/mL; pure substances: 0.01–100 µM). Budesonide (1 nM) served as a positive control for anti-inflammatory activity. Concomitantly, 100 ng/mL LPS was applied as inflammatory stimulus.
3
Characterization of Particles in Rat Alveolar Macrophages
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The biological characterization of the particles was performed with the cell line NR8383 (rat alveolar macrophages, LGC Standards GmbH, Wesel, Germany). The cells were cultivated with Ham's F-12 medium containing 15% fetal calf serum (FCS, GIBCO, Invitrogen, Karlsruhe, Germany) in 175 cm2 cell culture flasks (BD Falcon, Becton Dickinson GmbH, Heidelberg, Germany) at standard cell culture conditions (humidified atmosphere, 37 °C, 5% CO2). NR8383 cells were partly adherent and partly non-adherent. The ratio between adherent and non-adherent cells was about 1:1. For cell experiments, adherent cells were detached from the cell culture flasks with a TPP cell scraper (TPP Techno Plastic Products AG, Trasadingen, Switzerland), subsequently combined with non-adherent cells and seeded into 24-well cell culture plates (BD Falcon) at a concentration of 2.4 × 105 cells cm−2.
4
HUVEC Tube Formation Assay with Ang-2 Inhibition
5
IPEC-J2 Cell Culture Protocol
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The Intestinal porcine epithelial cell (IPEC-J2) model was a kind gift from Dr. Nicholas Gabler (Iowa State University, Ames, IA). Cells were cultured in a humidified incubator at 37°C under 5% CO2 in 25 cm2 cell culture flask (Corning Inc., Corning, NY). Cells were grown in Dulbecco’s modified Eagle’s medium: Nutrient Mixture F-12 (DMEM/F12; Sigma-Aldrich, St. Louis, MO) with 5% fetal bovine serum (FBS; Mediatech. Inc., Manassas, VA) supplemented with 1% insulin-transferrin-selenium premix (ITS premix: human recombinant insulin 1 mg/ml, transferrin 0.6 mg/ml and selenium 0.6 μg/ml; Corning Inc., Corning, NY), 5 ng/ml epidermal growth factor (EGF: Sigma-Aldrich, St. Louis, MO), and 1% penicillin-streptomycin mixture (Mediatech. Inc., Manassas, VA). Cells were seeded into 24-well cell culture plates (BD Falcon, Corning Inc., Corning, NY) at 0.5 × 105 cells/ml to form a confluent monolayer within 4 days, and then switched to the same medium without FBS to induce differentiation. Medium was replaced every 2 days, and cells were challenged with LPS on day 7 post-differentiation.
6
DENV-2 Infection in HUVEC Senescence
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HUVECs at young, intermediate young, early senescent and late senescent were seeded into 24-well cell culture plates (Falcon, BD Biosciences, USA) at a density of 5 x 104 cells/well. The following day, the cell culture medium was removed and passage 3 clinical isolate of DENV type-2 (DENV-2) strain MY91-99133 (Department of Medical Microbiology, University Malaya Medical Centre Virology repository) was added to give an estimated multiplicity of infection (MOI) of 10 per cell. Cells were incubated with the inoculum for 1 hour at room temperature with constant gentle agitation to allow virus adsorption. After an hour, the inoculum was removed and fresh ECM with 2% FBS was added. Cells were cultured for 7 days at 37ºC in a humidified incubator with 5% CO2. The cell culture supernatant was centrifuged and used for virus infectivity or replication assay by focus-forming assay and quantitative real-time (RT)-polymerase chain reaction (PCR). The remaining cell monolayers were trypsinized and stained for β-gal staining and cell cycle analysis.
7
Rearing Honeybee Larvae under Controlled Conditions
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Apis mellifera larvae were collected from nine hives near Fargo, Cass County, North Dakota during a three-week period in the summer of 2015. Hives were supplemented with pollen patties (Mann Lake, MN, USA) and a 1 : 1 sucrose–water solution (Brushy Mountain Bee Farm, NC, USA) during poor foraging conditions. First instar larvae (0–21 h old) were transferred into 24-well cell culture plates (Falcon, Corning, Durham, NC) and placed onto 10 µl of diet. The 24-well plates were stored inside a modulator incubator chamber (Billups-Rothenberg, del Mar, CA, USA). Larvae were kept at a constant 34°C, darkness and relative humidity (RH) of 96% using potassium sulfate (K2SO4) [33 (link)]. Larvae were fed according to treatment in a factorial design of nine diet qualities and eight quantities with an additional ad libitum treatment for the medium diet, as described in the following sections. At the prepupal stage they were moved into 24-well cell culture plates containing Kimwipes (Kimtech Science, USA) sterilized in EtOH [34 ]. Pupae were maintained at a constant 34°C, darkness and 75% RH using NaCl until adult eclosion. Adults were stored at −20°C.
8
Culturing Rat Alveolar Macrophages
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The biological effect of the particles was studied with the cell line NR8383 (rat alveolar macrophages, LGC Standards GmbH, Wesel, Germany). The cells were cultivated in Ham’s F12 medium containing 15% fetal calf serum (FCS, GIBCO, Invitrogen, Karlsruhe, Germany) in 175 cm2 cell culture flasks (BD Falcon, Becton Dickinson GmbH, Heidelberg, Germany) at standard cell culture conditions (humidified atmosphere, 37 °C, 5% CO2). The NR8383 cells were only partly adherent. The ratio between adherent and non-adherent cells was about 1:1. For cell experiments, adherent cells were detached from the cell culture flasks with a TPP cell scraper (TPP Techno Plastic Products AG, Trasadingen, Switzerland), subsequently combined with non-adherent cells, and seeded into 24-well cell culture plates (BD Falcon) at a concentration of 2.4 × 105 cells cm−2.
9
Cultivation of Bone Marrow Mesenchymal Stem Cells
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Bone marrow derived human mesenchymal stem cells (BM-hMSCs; 5 th to 10 th passage, Lonza, Basel, Switzerland) served as control and were cultivated in cell culture medium RPMI1640 (GIBCO, Invitrogen) containing 10% (v/v) fetal calf serum (FCS; GIBCO, Invitrogen) and Lglutamine (0.3 g L -1 ; GIBCO, Invitrogen) using 75 cm 2 culture flasks (BD Falcon). Cells were grown at 37 °C in a humidified 5% CO 2 atmosphere and sub-cultivated every 7 to 14 d depending on cell proliferation. After washing with PBS, growing hMSC were detached from the culture flasks by addition of 0.2 mL cm -2 0.25% trypsin/0.05% EDTA for 5 min at 37 °C. Subsequently, cells were harvested, washed twice with RPMI/FCS and seeded at a density of 1.5 x 10 4 cells per well in 24-well cell culture plates (BD Falcon).
10
Caco-2 Cell Permeability Assay
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Transparent polyethylene terephthalate inserts (0.4 μm) and 24-well cell culture plates were obtained from BD Biosciences Co. (San Diego, CA, USA). For assessment of in vitro ENCC permeability, Caco-2 cell suspensions (300 μL; 1×106 cells/mL) was plated onto transwell inserts (in the apical compartment) to form a cell monolayer and 900 μL of the culture medium was subsequently added to the lower side (basolateral layer). The culture medium was changed every 2 to 3 days until the cells were fully differentiated, and the transepithelial electrical resistance (TEER) value was measured every day to confirm the formation of the in vitro intestinal monolayer. When the TEER reached 500 Ωcm2, curcumin samples were added to the apical compartments, and the culture medium from both compartments were collected to determine curcumin concentrations at different time points (30, 60, and 90 min).
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