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6 well plate

Manufactured by Celltreat
Sourced in China, United States

The 6-well plate is a laboratory item used for cell culture and experimentation. It consists of a flat, rectangular base with six individual wells, each designed to hold a specific volume of liquid or cell sample. The 6-well plate is a commonly used tool in various scientific and research applications.

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6 protocols using 6 well plate

1

Live Cell Imaging and Cell Fate Tracking

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Two days prior to microscopy cells were passaged and seeded in a 6-well plate (Celltreat, China), at roughly 50 to 80% confluence. Live cell imaging was performed using the Marianas Live Cell system based around a Zeiss Axiovert 200M microscope stand, and the SlideBook6 software (Intelligent Imaging Innovations, Inc, Denver, CO.). Images were collected every 10 minutes for 24 hours with 10X objective lens magnification. Once the live cell microscopy was completed, the captured images were loaded into SlideBook Reader Software (Intelligent Imaging Innovations). Under each condition, one hundred cells were manually tracked for cell fates in the experiment. Cell behaviors were entered into Microsoft Excel Spreadsheet to generate cell profile graphs, as illustrated in a previous study [38 (link)] (Figures S1 and S2). Statistical significance was analyzed using an unpaired 2-tailed Student's t-test. The values are presented as the means ± standard errors. A p-value < 0.05 was considered statistically significant.
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2

Subcellular Fluorescence Imaging Protocol

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The subcellular fluorescence experiments were performed by culturing the cells in a 6-well plate (Celltreat) with 10 μM concentration compound solution at 37 °C for 6 h. Afterwards, the medium was removed and cells were washed with 1X PBS three times. Images were acquired using a Leica DMRXA2 microscope with a water immersion objective and DAPI, GFP, and TRITC filter cubes (Chroma Technologies).
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3

Recellularization of Decellularized Bronchi

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Decellularized bronchi were split in two and secured on a sterile support, then cut into small pieces of a maximum of 1x1cm and rinsed in sterile PBS 1X. Tissues were then placed in a 24-well plate (Costar, Washington, D.C.) and recellularized with the ASM cells between P4 and P7 at a concentration of 158,000 cells/cm 2 . One and a half milliliters of medium were added to the culture under the same condition described above. After a 48-hour incubation, allowing primary adhesion, 1 ml of the medium was changed in each well. Then, the medium was changed every other day. Tissues were maintained in culture, in the same well, between 48 hours and 41 days or transferred at 31 days to a 6-well plate (Celltreat, Pepperell, MA) for 10 more days. Samples were collected at day 2, 7, 14, 21 and 31 without tissue transfer and at day 41 with and without tissue transfer (Supplemental figure 1).
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4

MMP1 Effect on Biofilm Formation

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Established biofilms were first grown as follows. 300 μl of log-phase culture inoculation was added to 5 ml BHI media in wells of untreated 6-well plate (Celltreat Scientific Products, 229506), where biofilms were grown for 3 to 7 days by immersing 22 mm × 22 mm plastic coverslips (Carolina Biological Supply Company, 632900) in the solution. To study the effect of MMP1, 500 μl of 1 mg/ml MMP1 was added in each well. For control experiments, 500 μl of BHI media and protein buffer were added instead of MMP1. To image biofilms, the solution was aspirated and coverslips were washed three times with 500 μl of sterile PBS and dried at 37°C for 24 hr before imaging using a Phenom Pro-Scanning Electron microscope.
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5

Polystyrene Microparticle Uptake in U-2 OS Cells

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U-2 OS cells were cultured and plated in a 6-well plate (Celltreat, Pepperell, MA, USA). Once adherent, cells were treated with 5 μg/mL of 0.1 μm PS-MPs or 20 μg/mL of 2 μm PS-MPs for 24 h. After exposure, the cells were washed with sterile PBS (Gibco, Waltham, MA, USA), detached, centrifuged in DMEM (Gibco, Waltham, MA, USA) at 1000× g at 4 °C for 5 min to remove excess PS-MPs, and re-plated on 13 mm coverslips in a 6-well plate. After 24 h, the cells were washed 3 times with PBS, fixed for 10 min with 4% formaldehyde at RT in PBS, permeabilized with 0.1% Triton X-100 (Sigma, St. Louis, MO, USA) in PBS at RT for 15 min, stained with Hoescht (1:2000, H1399, Invitrogen, Waltham, MA, USA) and Phalloidin (1:500, PF7501, EPM Scientific, New York, NY, USA) for 5 min at RT, washed 3 times in PBS, and mounted onto glass slides with an aqueous mounting medium. Fluorescence imaging (Leica THUNDER DMi8 3D Fluorescence Imaging System, Leica Biosystems, Wetzlar, Germany and LAS X 3D Analysis Software v. 2018.7.3, Leica Biosystems, Wetzlar, Germany) was used to identify the dyed red fluorescent polystyrene particles using a TRITC (550 nm) filter.
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6

TLR7 Expression in RAW264.7 Cells

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RAW264.7 cells were cultured at high and low density (2500 and 400 cells/mm2 respectively) overnight in a 6-well plate (CellTreat 229106) in DMEM media supplemented with 10% FBS. Cells at each density were divided into five aliquots of equal cell count (approximately 0.5 x 106) and washed with PBS, stained with a live dead reactive dye (BioLegend L34975), fixed and permeabilized with Cyto-Fast Fix/Perm Kit (BioLegend 426803) following kit instructions, and then blocked with anti-mouse CD16/32 antibody (BioLegend 101319) at 10 ug/mL. For each density, three samples were stained with PE anti-mouse CD287 (TLR7) Antibody (BioLegend 160003) at 5 ug/mL, one with PE Mouse IgG1, κ Isotype Ctrl (FC) Antibody (BioLegend 400113) at 5 ug/mL, and one left unstained, at the steps indicated in the Fix/Perm protocol. The cells were then washed and resuspended in Cell Staining Buffer (BioLegend 420201) and run on an ACEA NovoCyte Flow Cytometer.
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