The epithelial cells and components used in the 3D cultures of the breast used for validation of SAMA have been previously described [11 (link)]. Briefly, cells were suspended in a final volume of 1.5 mL of collagen (1 mg/ml) and poured into 12-well plates (Becton Dickinson). The mixture was allowed to congeal for 30 min at 37°C and 1.5 mL of the CD-FBS or CD-FBS medium containing estradiol alone (10−10,10−9M), or in combination with promegestone (10−10M), or prolactin (10−7M)was added to each well. Gels were detached [10 (link)] and cultures were maintained for two weeks for cell organization analysis and the medium was changed every two days. After two weeks, gels were fixed overnight in 10% phosphate-buffered formalin and stained with Carmine-alum.
12 well plate
Manufactured by BD
Sourced in United States, Japan, Germany
12-well plates are a type of multi-well cell culture plate used for various laboratory applications. Each plate contains 12 individual wells, providing a platform for simultaneous experiments or sample cultivation. The plates are typically made of durable, tissue culture-treated polystyrene material, ensuring optimal cell adherence and growth conditions.
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Lab products found in correlation
6 well plate, by BD (5 mentions)
Cellstart, by Thermo Fisher Scientific (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Poly l lysine (pll), by Merck Group (4 mentions)
Transwell insert, by Corning (4 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Stemprosfm cts, by Thermo Fisher Scientific (3 mentions)
Phenol red, by Merck Group (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Zen blue software, by Zeiss (2 mentions)
H2dcfda, by Thermo Fisher Scientific (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Telaval 31 light microscope, by Zeiss (2 mentions)
Puromycin, by Thermo Fisher Scientific (2 mentions)
Polybrene, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
126 protocols using 12 well plate
1
3D Breast Culture Validation Protocol for SAMA
2
Pancreatic CSCs Uptake of Mang-NPs
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Pancreatic CSCs and cell lines were grown in 12-well plates (Beckton Dickinson, Bedford, MA) and treated with or without a mixture of coumarin-6 containing Mang-NPs (5 μM) and Hoechst33342 (1 mg/ml) for various time points (0–24 hrs). After washing with PBS, stained cells were visualized under a fluorescent microscope.
3
Coculture of HepG2 Cells with BMSCs for Steatosis Induction
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The human hepatoblastoma cell line HepG2 cells were purchased from Summit Pharmaceuticals International (Tokyo, Japan). HepG2 cells were cultured in DMEM (Thermo Fisher Scientific) supplemented with 10% FBS (Thermo Fisher Scientific) and 100-µg/ml gentamicin (Thermo Fisher Scientific). HepG2 cells were seeded onto 12-well plates (Becton Dickinson Labware, NJ). To induce steatosis, HepG2 cells were exposed to 0.5-mM free fatty acid (FFA) mixture (oleic acid/palmitic acid, 2:1) (Sigma-Aldrich, St. Louis, MO) for 24 h. HepG2 cells were divided into the following two groups: 1) FFA-treated group; and 2) FFA and BMSC co-culture group. Then, 5 × 104 BMSCs were seeded onto the 0.4-µm-pore size Cell Culture Insert (Becton Dickinson Labware) and placed into the 12-well plate with the HepG2 cells that were initially seeded. In the transwell system, only secretome from BMSC cultured in the upper compartment could pass through the membrane to contact with HepG2 cells cultured in the lower compartment. After another 48 h, the cells were collected for Oil-red O staining (Sigma-Aldrich) and thereafter, incubated for 5 min in 100% isopropanol (Wako). The Oil-red O staining solution was then extracted from each well, and absorbance at a wavelength of 492 nm was measured using the Infinite M 200Pro (Tecan, Kanagawa, Japan).
4
3D Culturing of Breast Epithelial Cells
5
Measuring TLR4-Induced IL-8 in VFF
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To determine the direct downstream signaling effect after TLR4 activation in VFF, we measured the production of IL-8 pro-inflammatory cytokine after LPS stimulation. VFF were seeded at near confluence 2 × 105 per well into 12 well plates (Becton Dickinson Labware) to control for growth kinetics. Following 24 hrs of serum starvation, DMEM-10% FBS supplemented with or without 5 μg/mL LPS was added to each well. After 8 hrs incubation, supernatants were removed and stored at −80°C. Protein secretion levels were measured using an IL-8 ELISA according to the manufacturer’s protocol (Invitrogen, USA). Standard curves were used to measure the quantity of each sample. Non-stimulated cells and U937cells stimulated with and without LPS were used as controls.
6
Cell Culture of Dopaminergic, Astroglial, and Microglial Lines
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Dopaminergic N27 cell line derived from rat female mesencephalic tissue (SCC048, Millipore, MA, USA) was cultured in RPMI 1640 medium supplemented with 10 % FBS, 2 mM L-glutamine (Sigma), 100 U/ml penicillin, and 100 μg/ml streptomycin.
The C6 astroglial cells (CB_92090409, Sigma) were cultured in Ham’s F12 medium with 10% FBS, 2 mM L-glutamine (Sigma), 100 U/ml penicillin and 100 μg/ml streptomycin.
The HMC3 microglial cell line, a gift from Dr. Dora Brites (Research Institute for Medicines, University of Lisboa, Portugal), established through SV40-dependent immortalization of a human fetal brain-derived primary microglial culture, was cultured in DMEM medium (Sigma) with 2 mM L-glutamine (Sigma), 100 U/ml penicillin and 100 μg/ml streptomycin.
All cell line cultures were maintained at 37 °C and 5% CO2 in a humidified incubator in a 75 cm2 culture flask. Once cells became confluent, they were re-seeded onto 12-well plates (Falcon, Becton Dickinson, Franklin Lakes, NJ, USA), containing glass coverslips, at a density of 0.5 × 105 cells/cm2
The C6 astroglial cells (CB_92090409, Sigma) were cultured in Ham’s F12 medium with 10% FBS, 2 mM L-glutamine (Sigma), 100 U/ml penicillin and 100 μg/ml streptomycin.
The HMC3 microglial cell line, a gift from Dr. Dora Brites (Research Institute for Medicines, University of Lisboa, Portugal), established through SV40-dependent immortalization of a human fetal brain-derived primary microglial culture, was cultured in DMEM medium (Sigma) with 2 mM L-glutamine (Sigma), 100 U/ml penicillin and 100 μg/ml streptomycin.
All cell line cultures were maintained at 37 °C and 5% CO2 in a humidified incubator in a 75 cm2 culture flask. Once cells became confluent, they were re-seeded onto 12-well plates (Falcon, Becton Dickinson, Franklin Lakes, NJ, USA), containing glass coverslips, at a density of 0.5 × 105 cells/cm2
7
Coumarin-6 Mang-NPs Uptake in Colorectal Cells
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Colorectal cancer cells were grown in 12‐well plates (Beckton Dickinson, Bedford, MA) and treated with or without a mixture of coumarin‐6 containing Mang‐NPs (5 µmol/L) and DAPI (1 mg/mL) for various time points (0‐24 hours). After washing with PBS, stained cells were visualized under a fluorescent microscope.
8
Evaluating Durability and Protein Expression of PRGF Gel
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To evaluate the durability of the produced gel and to analyze the protein expression of the MNC in the PRGF, pieces of SIS (0.9 cm2) were placed in 8-chamber slides (Merck Millipore, Cork, Ireland) and 350 μL PRGF was added on top and incubated for 30–45 min in room temperature until gel was formed (Table 1 ). PRGF (1 mL) without SIS was produced as a control. The gel pieces and the PRGF-SIS constructs were transferred to 12-well plates (Becton-Dickinson) and 350 μL RPMI 1640 medium (Life Technologies) supplemented with 10% pooled inactivated human AB serum, 100 U/mL penicillin, and 100 mg/mL streptomycin (HyClone Laboratories, South Logan, UT, USA), and 20 mM L-glutamine (Invitrogen) was added per well. At days 0, 3, 7, 10, and 14, the PRGF gel and PRGF-SIS constructs were collected, washed in PBS, and transferred to 4% PFA for 3 days and thereafter paraffin embedded and sectioned. The sections were stained as above with HTX/eosin for morphology, CD34 and CD45 (both hematopoietic surface markers, 1 : 100, Dako) for surface epitope expression, Ki67 (1 : 500, Life Technologies), and for activated caspase 3 (1 : 250, Invitrogen).
9
Silencing PHB, PHB2, and ILK1 in Cancer Cells
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The MDA-MB-231 human breast cancer cell line and PANC1 human pancreatic cancer cell line were transiently transfected using siRNA against PHB (siGENOME Human PHB (5254) siRNA – SMART pool; Dharmacom), PHB2 (siGENOME Human Phb2 (11331) siRNA – SMART pool; Dharmacon), ILK1 (siGENOME Human Ilk (3611) siRNA – SMART pool; Dharmacon), and control (siGENOME Non-Targeting siRNA Pool; Dharmacon) using DharmaFECT1 transfection reagent (#T-2001-02; Dharmacon) for 72 h. For validation of the transfection efficiency, western blot analysis was employed. In a wound healing assay, cells were plated in 12-well plates (#3043, Becton Dickinson) and cultured. After transfection, a narrow gap was created with a pipette tip in the confluent cell monolayer and monitored for 24 h for recolonization.
10
Chronic and Acute LPS Stimulation of Monocytes
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For chronic LPS stimulus, monocytes were placed in polytetrafluoroethylene inserts (Millipore, Carrigtwohill, Ireland) within 12-well plates (Becton Dickinson, Franklin Lakes, NJ) at 3 × 106 per mL for 16 hours at 37°C ± LPS (1 μg/mL). For acute LPS stimulus, cells were harvested from tissue plates and stimulated as previously described.
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