Primary cultures of astrocytes were prepared as previously described (Hertz et al., 1998 (link)). The neopallia of the cerebral hemispheres of newborn CD-1 mice were aseptically isolated, vortexed to dissociate the tissue, filtered through nylon meshes with pore sizes of 80 μm and subsequently 10 μm, diluted in culture medium, and planted in Falcon Primaria culture dishes. The culture medium was a Dulbecco’s Minimum Essential Medium (DMEM) with 7.5 mM glucose, initially containing 20% horse serum, and the cultures were incubated at 37°C in a humidified atmosphere of CO2/air (5:95%). The culture medium was exchanged with fresh medium of similar composition on day 3, and subsequently every 3–4 days. From day 3, the serum concentration was reduced to 10%, and after the age of 2 weeks, 0.25 mM dibutyryl cyclic AMP (dBcAMP) was included in the medium, leading to morphological and functional differentiation (Meier et al., 1991 (link)). The cultures were used after at least 3 weeks of culture.
Primaria culture dishes
Manufactured by BD
Sourced in United Kingdom
Primaria culture dishes are sterile, tissue culture-treated polystyrene dishes designed for cell and tissue culture applications. They provide a consistent and optimized surface for the attachment and growth of a variety of cell types.
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Lab products found in correlation
1640 medium, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Pen strep solution, by Thermo Fisher Scientific (1 mentions)
Gm csf, by R&D Systems (1 mentions)
Dulbecco modified eagle medium (dmem), by Fujifilm (1 mentions)
Axiovert 200m microscope, by Zeiss (1 mentions)
Axiovision software, by Zeiss (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Collagenase 2, by Worthington (1 mentions)
Automacs, by Miltenyi Biotec (1 mentions)
18 hepe, by Cayman Chemical (1 mentions)
6 protocols using primaria culture dishes
1
Astrocyte Primary Culture Protocol
2
Isolation and Differentiation of Human Monocytes
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Primary human monocytes were seeded in Primaria culture dishes (BD Falcon, BD Biosciences, Oxford, UK) at 3 × 105 cells/cm2, and maintained in Roswell Park Memorial Institute medium 1640 (Gibco, Life Technologies); 10% foetal bovine serum (Gibco); 2 mM L‐glutamine (Gibco, Life Technologies); 5 U/mL penicillin and 5 μg/mL streptomycin (Gibco PenStrep solution) in 5% CO2 atmosphere at 37°C. Pre‐testing has shown that this protocol produces monocyte cultures of at least 95% CD14+ monocytes and these were used in experiments from 1 to 3 days in vitro (DIV). For some experiments, monocytes were differentiated into macrophages with the addition of 20 ng/mL granulocyte macrophage colony‐stimulating factor (GM‐CSF, R&D Systems, Oxford, UK) to the culture medium on seeding. Cells were given a complete media change with addition of fresh GM‐CSF at 3 DIV and have been confirmed by factor analysis to be fully differentiated macrophages at 6 DIV and were used in experiments at this stage.
3
Isolation and Culture of Lung Fibroblasts
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Lungs were perfused and digested with collagenase II (Worthington Biochemical Crop). Dissociated cells were incubated for an hour. The remaining cells were resuspended in DMEM (Wako) containing 10% fetal bovine serum and placed on Primaria culture dishes (BD). After 24 h, the culture medium was changed. After further 3 days and 5 days, the fibroblasts cultures were passaged, and used in the experiments.
4
Culturing Primary Human Corneal Stromal Fibroblasts
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Human donor cornea-sclera rims were obtained from Miami Eye Bank (Miami, FL) and Saving Sight (Kansas City, MO). Central cornea was excised using an 8.0 mm trephine, and corneal epithelial and endothelial were removed by light scraping using a Beaver #64 blade. Remaining cornea stroma was cut into small 1–2 mm pieces and placed onto 35 mm Primaria Culture dishes (BD Biosciences, Franklin Lake, NJ). DMEM/F-12 with 10% FBS, 1X ITS, 1% pen/strep and 0.1% AmphoB was used to maintain explant cultures in 37 °C with 5% CO2 till confluence. Explant pieces were removed carefully using sterile forceps and pHCSFs were expanded up to 4 passages. Media with 5% FBS was used to maintain subsequent passages. Passage 2 pHCSFs were used for all in vitro experiments. Unless otherwise stated, cultures were maintained for 3 days to reach 80% confluence before drug treatments.
5
NF-κB Activation in RAW264.7 Macrophages
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RAW264.7 macrophage cells stably transfected with an NF-κB-GFP reporter construct were used similarly to our previous work [23 ,58 ]. For experiments, the reporter cells were distributed into 24-well Primaria culture dishes (Primaria, BD Biosciences) at 2.5 x 105 cells per well. Wells were treated with media, Escherichia coli lipopolysaccharide (LPS), or strains of LVS. Twenty-two hours post-infection, wells were washed with Hank’s Balanced Salt Solution and GFP fluorescence was visualized on a Zeiss Axiovert 200M microscope. Images were collected by using the Zeiss Axiovision software, converted to tiff files using ImageJ, and the brightness and contrast were adjusted consistently across all images using Microsoft Powerpoint.
6
Isolation and Co-culture of Cardiac Fibroblasts
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Hearts were perfused and digested with collagenase II (Worthington Biochemical Corp). Dissociated cells were incubated with anti-CD45-conjugated microbeads (Miltenyi Biotec), followed by selective depletion of CD45+ cells using autoMACS (Miltenyi Biotec). The remaining CD45- cells were resuspended in DMEM (Life Technologies) containing 10% fetal bovine serum and placed on Primaria culture dishes (BD). After 2 h, the culture medium was changed to remove any myocytes and endothelial cells still weakly attached to the dish. After 24 h, the culture medium was changed again, and the fibroblast cultures were grown to 85% confluence, at which point the medium was replaced with DMEM containing 0.1% FBS (serum-reduced medium). After a further 24 h, the isolated fibroblasts were cultured with co-cultured macrophages and macrophage-conditioned medium with or without 18-HEPE (Cayman Chemical).
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