The FACS analysis was performed as described previously [36 (link)]. BMMs (1 × 106 cells/well, n = 4 per group) were seeded into six-well culture plates (Falcon) and treated with each concentration of MP (1 μg/mL) and TEGO (100 μg/mL) containing LPS (0.1 μg/mL). After 24 h, cells were fixed with 4% paraformaldehyde and incubated with primary antibodies, including anti-CD68 (pan-macrophage marker), anti-CD86 (M1-phenotype marker), and anti-CD206 (M2-phenotype marker) antibodies for one hour. After washing, BMMs were stained with Alexa 488 and Alexa 647. The marker expression was analyzed on a CytoFLEX Flow Cytometer (CytoFLEX V5-B5-R3, Beckman Coulter, Brea, CA, USA), and data were analyzed using the CytExpert software 2.4 (Beckman Coulter).
Six well culture plates
Manufactured by BD
Sourced in United States
Six-well culture plates are a type of laboratory equipment designed for cell culture applications. They provide a standardized platform for growing and maintaining cells in a controlled environment. Each plate features six individual wells, allowing for multiple experiments or conditions to be tested simultaneously.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Sas software, by SAS Institute (2 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
Cytexpert software 2, by Beckman Coulter (1 mentions)
Fortecortin, by Merck Group (1 mentions)
Human insulin, by Sanofi (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
5 protocols using six well culture plates
1
Macrophage Polarization Dynamics by FACS Analysis
2
2D Hepatocyte Culture Protocol
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In parallel to bioreactor cultures, experiments with cells from the same donor were performed in 2-D plates for comparison. Six-well culture plates (Falcon; BD Biosciences, San Jose, CA, USA) were coated with rat-tail collagen prepared according to a protocol described previously.17 Freshly isolated human hepatocytes were suspended in Williams Medium E with GlutaMax (Thermo Fisher Scientific, Waltham, MA, USA), containing penicillin–streptomycin (100,000 U/L–100 mg/L), 15 mM HEPES, 1 mM sodium pyruvate, 1% non essential amino acids, 10% FBS (all Thermo Fisher Scientific), 0.8 mg/L dexamethasone (Fortecortin; Merck, Darmstadt, Germany), and 1 mM human insulin (Sanofi Aventis, Frankfurt am Main, Germany), referred to as “full medium”. Cells were seeded in the collagen-coated plates at a density of 1.4 million cells per well (Figure 3B and D ). From 24 hours onward, the culture medium was exchanged daily with the same medium, but without FBS and hormones (starving medium).
3
Quantifying Inflammatory Cytokine Responses
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BMMs (1 × 106 cells/well, n = 4 per group) were seeded into six-well culture plates (Falcon) to perform the qRT-PCR analyses. Cells were treated with each concentration of MP (1 μg/mL) and TEGO (100 μg/mL) containing LPS (0.1 μg/mL). After 24 h, total RNA from seeded cells were extracted using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. The qRT-PCR analyses were performed as described previously [8 (link),33 (link),34 (link)]. The primers were obtained from Bioneer (Daejeon, Republic of Korea). The relative gene expression values of IL-6, IL-1β, TNF-α, IL-4, IL-10, and TGF-β were normalized to those of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) by using the 2-ΔΔCT calculation [37 (link)]. A detailed description of the qRT-PCR method, including the nucleotide sequences of primers, is provided in the Supporting Information .
4
Cerebellar Neuron Cultures and Antioxidant Assay
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Primary cultures of cerebellar neurons were prepared from P5 C57/BL6 mice. Six well culture plates (Falcon, Indianapolis, IN) were coated with poly-D-lysine and seeded with cerebellar neurons at a density of 1.5 × 106 cells/well, and maintained with DMEM media supplemented with L-glutamine, pen-strep, 30% sucrose, B-27, and N-2. Ara-C was added 24 hours after seeding to produce a homogenous neuronal culture. Cells were grown to confluence for 6 days, with half media changes every 48 hours. The negative control received no treatment, the vehicle was treated with 500 μM MG. Antioxidant treatment groups were treated for 24 hours by adding 500 μM MG first and followed by 10 μM flavonoid.
5
Nematode Infection Assay in Arabidopsis
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Arabidopsis thaliana (Columbia and Wassilewskija ecotypes) seeds were surface sterilized and transferred (one seed per well) into six-well culture plates (Falcon) containing 6 mL of sterile modified Knops medium [64 (link)] solidified with 0.8% Daishin agar (Brunschwig Chemie) as previously described [8 (link)]. Seeds on plates were placed in a 24 °C growth chamber under a 16 h light/8 h dark cycle for 2 weeks. Surface sterilized pre-J2 nematodes were suspended in 1.5% low-melting-point agarose to allow even distribution and to facilitate their movement into solid Knops medium. Plants were inoculated with approximately 60 J2 per plant and developed cysts (for sugar beet cyst nematodes) and galls (for root-knot nematodes) were counted 3–4 weeks post-infection, using a dissecting microscope. Nematode infection assays were conducted on two independent biological replicates, and similar results were obtained (the data of one experiment is presented). The means and standard errors of 18 replicates per treatment were calculated. Statistical differences were determined by the paired t-test with an alpha level of 0.05 using SAS software (Cary, NC, USA).
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