12 well plate

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12-well plates are a type of multi-well cell culture plate used for various laboratory applications. They provide 12 individual wells for the cultivation, testing, or analysis of cells or other biological samples. The wells are arranged in a 3x4 grid pattern and typically have a flat bottom design. 12-well plates offer a convenient format for parallel experiments or sample processing in a compact format.

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12-well microtiter plates (7 citations) 12-well polystyrene plates (2 citations) 12-well culture plates (2 citations) 12 well tissue culture plates (2 citations)

20 protocols using 12 well plate

Fluorescent Protein Visualization of CD Receptors

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The CDS of CD80/86, CD28, and CD152 were amplified by PCR using specifically designed primers (Table 1). The PCR products were digested and inserted into a pEGFP-N1 plasmid using the EcoR I and BamHI restriction enzymes. The pEGFP-CD80/86, pEGFP-CD28, and pEGFP-CD152 plasmids were transformed into Trans5α chemically competent cells (TransGen), which were subsequently cultured in LB Broth supplemented with 50 mg/L kanamycin for 12 hours at 37°C. The plasmids were then extracted from the cells using an E.Z.N.A. Plasmid Maxi Kit (Omega). HEK293T cells (5 × 10⁵ cells) were seeded into each well of a 12-well plate (VWR) and cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco) for 12 hours at 37°C in 5% CO₂. Then, either 1.6 µg of the pEGFP-N1, pEGFP-CD80/86, pEGFP-CD28, or pEGFP-CD152 plasmid was transfected into the HEK293T cells in each well using 4 µL TransIntro™ EL Transfection Reagent (TransGen), according to the manufacturer’s instructions. At 24 hours post-transfection, 4% paraformaldehyde (Biosharp) was added to the cells for 15 minutes to fix them. Afterward, the cells were stained with 100 ng/mL DAPI (Biosharp) for 15 minutes at room temperature. Finally, confocal microscopy (Nikon N-STORM) was used to obtain fluorescence images of the transfected HEK293T cells.

Lu T.Z., Liu X., Wu C.S., Ma Z.Y., Wang Y., Zhang Y.A, & Zhang X.J. (2022). Molecular and Functional Analyses of the Primordial Costimulatory Molecule CD80/86 and Its Receptors CD28 and CD152 (CTLA-4) in a Teleost Fish. Frontiers in Immunology, 13, 885005.

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Biofilm Formation on GNP/PDMS Composites

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To assess biofilm formation on GNP/PDMS composites, the composites were first sterilized through UV radiation for 1 h. Sterilized surfaces of PDMS, and 1, 2, 3, 4, and 5 wt% GNP/PDMS were placed on the microplate wells (12-well plate, VWR International, Carnaxide, Portugal) and inoculated with 3 mL of the bacterial suspension. Plates were incubated at 37 °C for 24 h under static conditions.

Oliveira I.M., Gomes M., Gomes L.C., Pereira M.F., Soares O.S, & Mergulhão F.J. (2022). Performance of Graphene/Polydimethylsiloxane Surfaces against S. aureus and P. aeruginosa Single- and Dual-Species Biofilms. Nanomaterials, 12(3), 355.

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Ultrasound-Triggered Dextran Release

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ULCs and 7% BIS microgels were loaded with 20 kDa dextran (VWR International, Radnor, PA, USA) labeled with fluorescein isothiocyanate (Fisher Scientific, Hampton, NH, USA) by swelling 10 mg of each microgel type in 500 μL of dextran solution (2.25 mg/ml dextran in 10 mM HEPES buffer, pH 7.4) for 24 hours. Microgel suspensions were spun in a microcentrifuge at 21.1 × g for 15 minutes. The supernatants were collected and the pellets were resuspended in 500 μL 10 mM HEPES, pH 7.4. Samples were centrifuged and supernatants were collected every hour for the first 6 hours and then at 12 and 24 hours. Samples exposed to ultrasound were transferred to a 12-well plate between collection time points (VWR International, Radnor, PA, USA) and kept in contact with a transducer applying ultrasound at a frequency of 1 MHz for 10 μs in cycles of 250 μs. Fluorescent readings from the supernatants collected at each time point were obtained using a plate reader. A standard curve was created using solutions of FITC-labeled dextran in 10 mM HEPES buffer. Cumulative drug release over 24 hours was calculated from the obtained fluorescence readings.

Joshi A., Nandi S., Chester D., Brown A.C, & Muller M. (2018). Study of poly (N-isopropylacrilamide-co-acrylic acid) (pNIPAM) microgel particle induced deformations of tissue mimicking phantom by ultrasound stimulation. Langmuir : the ACS journal of surfaces and colloids, 34(4), 1457-1465.

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PBMC Stimulation and Cytokine Profiling

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After thawing rapidly in a 37°C water bath, the PBMCs were plated at a density of 10⁶ cells/mL/well in a 12-well plate (VWR, Cat#: 10062-894), in glucose-free RPMI media, containing L-glutamine and 25 mM hepes (Gibco, Cat#: 11879020) supplemented with 10% FBS heat-inactivated (Gibco, Cat#: 10438026), 1% pen/strep (Gibco, Cat#: 15140122), and 1% sodium pyruvate (Corning, Cat#: 25-000-CI). The cells were stimulated in multiple batches, and each batch included a random sampling of approximately six participants. The PBMCs were stimulated with Escherichia coli O111:B4 lipopolysaccharides (LPS) (25 ng/mL, Millipore Sigma; 20 h) or αCD3/αCD28 Dynabeads (1 bead/cell; Gibco, Cat#: 11131D; 40 h) in a CO₂ incubator at 37°C prior to conditioned media collection. The unstimulated cells were incubated 40 h prior to conditioned media collection. The conditioned media was aliquoted and stored at −80°C for cytokine assays. The 20 and 40 h post-stimulation timepoints were determined based on prior studies.^{25-28 (link)}

Bachstetter A.D., Lutshumba J., Winford E., Abner E.L., Martin B.J., Harp J.P., Van Eldik L.J., Schmitt F.A., Wilcock D.M., Stowe A.M., Jicha G.A, & Nikolajczyk B.S. (2023). A blunted TH17 cytokine signature in women with mild cognitive impairment: insights from inflammatory profiling of a community-based cohort of older adults. Brain Communications, 5(5), fcad259.

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Myoblast Hypo-Osmotic Shock Monitoring

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WT (C25) and DYSF-null (AB320) human myoblasts were plated at 70% confluence on a 12-well plate (VWR) and transfected using Lipofectamine 2000 (Invitrogen) per manufacturer’s directions. After 24 h, myoblasts were washed with distilled water and hypo-osmotic shock was performed by incubating cells with distilled water. Cell fluorescence was followed with Fast imaging observer (Axio observer, Z1/×7, ×10 objective, ZEISS). Images were captured every minute for 30 min.

Ballouhey O., Courrier S., Kergourlay V., Gorokhova S., Cerino M., Krahn M., Lévy N, & Bartoli M. (2021). The Dysferlin Transcript Containing the Alternative Exon 40a is Essential for Myocyte Functions. Frontiers in Cell and Developmental Biology, 9, 754555.

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Breast Tumor Imaging with Nanoparticles

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For mouse 4T1 breast tumor production, a million tumor cells suspended in DPBS were transplanted into the mammary fat pad of female BALB/c mice orthotopically, as described previously [29 (link)]. After 14 days, mice were sacrificed under deep anesthesia and tumors were collected. Live 4T1 tumor slices were then sectioned at the thickness of 200 μm using a Leica VT1000P vibratome. Tumor slices were cultured in the 12-well plate (VWR, West Chester, PA, USA, Cat# 82050-930) and incubated with AgNP-555 or AgNP-555 + TP peptide in the FBS-free DMEM medium at 37 °C for 2 h. After incubation, the slices were etched, washed by DPBS, fixed with 4% PFA and mounted with the DAPI-containing mounting medium by coverslips. Nikon C2 confocal (Melville, NY, USA) was used to examine the tumor slices.

Li Y.X., Wei Y., Zhong R., Li L, & Pang H.B. (2021). Transportan Peptide Stimulates the Nanomaterial Internalization into Mammalian Cells in the Bystander Manner through Macropinocytosis. Pharmaceutics, 13(4), 552.

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Dosing TiO2 Nanoparticles in Lung and Cervical Cancer Cells

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A549 human lung epithelial carcinoma cells (CCL-185, ATCC, Manassas, VA) and HeLa human cervical carcinoma cells (CCL-2, ATCC) were cultured in Minimum Essential Medium (MEM, #61100, Invitrogen) supplemented with 10% fetal bovine serum (FBS, #10437028, Invitrogen) at 37 °C and 5% carbon dioxide. Cells were passaged with trypsin (#25200072, ThermoFisher Scientific) every 2–3 days. In T-25 flasks, TiO₂ NPs were used at a concentration of 800 μg mL⁻¹ for A549 cells with the working concentration determined by MTT assay. The ratio of NPs to cells was kept constant for all experiments and is reported throughout the text for experiments in well plates (12-well plates; #62406-165, VWR) and 35 mm optical dishes (#P35G-1.5-20-C, MatTek, Ashland, MA), based on the number of cells forming a confluent monolayer.

Jayaram D.T., Kumar A., Kippner L.E., Ho P.Y., Kemp M.L., Fan Y, & Payne C.K. (2019). TiO2 nanoparticles generate superoxide and alter gene expression in human lung cells. RSC Advances, 9(43), 25039-25047.

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Biofilm Formation Assay on PLA Surfaces

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Biofilm assays were performed on 12-well plates (VWR International, Carnaxide, Portugal) for 1 day at 5 °C under static conditions in order to mimic the conditions typically found in short-term food packaging. Before each experiment, the PLA film and the PLA-coated surfaces produced as described in Section 4.2 were sterilized by ultraviolet (UV) radiation for 30 min. Then, the sterilized surfaces were placed on the wells and inoculated with 3 mL of an overnight culture of E. coli SS2 GFP or P. putida in TSB adjusted to an optical density (OD) of 0.01 at 610 nm (1:10 dilution from an initial cell suspension at OD₆₁₀ nm = 0.1). The microplates were then kept at 5 °C for 1 h to promote bacterial attachment to the surface materials [36 (link),77 (link)]. After this adhesion step, the wells were emptied and refilled with 3 mL of sterile TSB, and the microplates were incubated to allow biofilm development. Furthermore, 3 mL of TSB was added to the wells containing sterilized surfaces to monitor their sterility throughout the experiments.
Biofilm formation experiments were performed in three independent assays, each one with three technical replicates.

Gomes L.C., Faria S.I., Valcarcel J., Vázquez J.A., Cerqueira M.A., Pastrana L., Bourbon A.I, & Mergulhão F.J. (2021). The Effect of Molecular Weight on the Antimicrobial Activity of Chitosan from Loligo opalescens for Food Packaging Applications. Marine Drugs, 19(7), 384.

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Cell Seeding and Transfection Optimization

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For adherent HeLa and HEK293T cells, 4 × 10⁵ cells were seeded directly into 12-well plates (VWR) for western blotting, and 1.5 × 10⁵ cells were seeded onto sterile coverslips (18 mm ø No. 1 German cover glasses, VWR VistaVision™, VWR International) deposited into 12-well plates for imaging. 0.8 × 10⁵ cells were seeded per chamber of 4-chamber wells (Lab-Tek®II Chambered #1.5 German Coverglass System; ThermoFisher) for live cell imaging microscopy. Cells were transfected 24 hr later with 2 µg plasmid DNA per well using JetPrime (PolyPlus) according to the manufacturer’s instructions. pcDNA3.1 was used as control for transfections with pNL4–3, and pRluc-N1 was used as control for experiments using p2-p1/Rluc (i.e., NC-RLuc). Jurkat T cells were transfected with 3 µg of plasmid DNA per 1 × 10⁶ cells using JetPrime (PolyPlus) for 12 days prior to treatments or collection.

Monette A., Niu M., Chen L., Rao S., James Gorelick R, & John Mouland A. (2020). Pan-retroviral Nucleocapsid-Mediated Phase Separation Regulates Genomic RNA Positioning and Trafficking. Cell reports, 31(3), 107520.

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Proliferation Assay for SK-MEL-28 and ASC Cells

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SK-MEL-28 and ASC were labeled (Table 1), trypsinized, and seeded with 5700 cells/cm² into 12-well plates (VWR International, Langenfeld, Germany). Proliferation was assessed on SK-MEL-28 using three technical replicates of each labeling concentration, on ASC using one well per donor (n = 3 independent donors). All well plates were incubated at 37 °C and 5% CO₂. Culture medium was changed daily. Cells were examined on days 1, 3, and 7. On each examination day, the cells to be assessed were washed with PBS and fixed using ice-cold 100% methanol (VWR International). Fixed cells were stored at 4 °C until analysis. Cell proliferation was quantified by 4′-6′-diamidino-2-phenylindole (DAPI) staining. The cells were stained using 1 mg/mL DAPI in PBS. DAPI fluorescence was analyzed by excitation at 365 nm and emission at 445/50 nm with a fluorescence microscope. Five images were taken following a specific layout with a magnification of 4×. Counting of DAPI-stained cell nuclei was done using the software CellProfiler3.1.9 [15 (link)].

Brune N., Mues B., Buhl E.M., Hintzen K.W., Jockenhoevel S., Cornelissen C.G., Slabu I, & Thiebes A.L. (2023). Dual Labeling of Primary Cells with Fluorescent Gadolinium Oxide Nanoparticles. Nanomaterials, 13(12), 1869.

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