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6 well polystyrene plates

Manufactured by Corning
Sourced in United States

The 6-well polystyrene plates are a type of laboratory equipment used for various cell culture applications. The product consists of a clear, flat-bottom plate with six individual wells, each designed to accommodate cell growth and experimentation. The plates are made of polystyrene, a commonly used material in the life sciences industry due to its optical clarity and compatibility with cell cultures. The core function of these plates is to provide a standardized, multi-well format for culturing and analyzing cells in a controlled environment.

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9 protocols using 6 well polystyrene plates

1

Dual-Species Biofilm Characterization

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Experiments were conducted using 8-well glass slide (Lab-Tek), polystyrene 6-well plates and 24-well plates (Corning). Spn and Sau strains were inoculated at a density of ~1 × 106 cfu/ml each in THY and incubated at 37°C in a 5% CO2 atmosphere for the indicated time. Control wells were only inoculated with Spn or Sau. Where indicated, bovine liver catalase (Sigma) was added to a final concentration of 1000 U/ml. Planktonic cells were removed, diluted and platted onto BAP or BAP with gentamicin to obtain cfu/ml for Spn or onto LBA or SMA to obtain cfu/ml of Sau. Biofilms were washed once with PBS, mixed with with 1 ml of sterile PBS and sonicated for 15 s in a Bransonic ultrasonic water bath (Branson, Danbury, CT), followed by extensive pipetting to remove remaining attached biofilm bacteria. Biofilms were diluted and platted as above.
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2

Biofilm Formation Assay Protocol

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For biofilm formation, mid-exponential planktonic cultures (2 ml×108 cells) were used to inoculate individual wells of polystyrene 6-well plates (Corning Incorporated, Costar, U.S.A.), and all wells supplemented with 2 ml BHI diluted 1∶5 with non-pyrogenic sterile dH2O. Cultures were incubated under static conditions at 37°C/5% CO2 with replacement of warm, fresh diluted BHI daily.
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3

Osteogenic Differentiation on Titanium Substrates

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Cells were cultured in 6-well polystyrene plates (Corning Inc., Corning, NY). Each ps-Ti well was coated with poly 2-hydroxyethyl methacrylate (Sigma-Aldrich, St Louis, MO) to ensure growth of cells on the titanium discs and to prevent cell migration onto polystyrene. Standard two-dimensional tissue culture polystyrene (tc-PS) wells were used in parallel for comparison with ps-Ti experiments. To accomplish adsorption, cell suspensions of approximately 2.5 × 105 cells in 500 μL were applied to either the surface of ps-Ti discs (placed in the well of a 6-well plate) or tc-PS wells. An additional 500 μL of medium was added to the bottom of each well to prevent desiccation of the cells during adsorption. After 2-3 hours of adsorption in a standard incubator (37° C and 5% CO2), 3 mL of media were added to each well (day −1). Following adsorption, the cells were incubated for 24 h, and maintenance medium was replaced with osteogenic medium (day 0). Osteogenic media changes were performed every 3-4 days (i.e., twice weekly) throughout the 28-day duration of the study (Table 1; Supplementary Figure 1).
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4

Western Blot Protein Expression Analysis

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For Western blots, cells were transfected on 6-well polystyrene plates (Corning). 24 h after transfection, the cells were washed twice with ice-cold PBS followed by scraping and extraction on ice for 30 min in a buffer containing 10 mM Tris-HCl, pH 6.8, 1 mM EDTA, 150 mM NaCl, 0.25% Nonidet P-40, 1% Triton X-100, 1 µM NaF and protease inhibitor mixture tablets (Roche Applied Science). Cell debris was removed by centrifugation at 16,000× g. The protein concentrations were determined using the BCA protein assay kit (Pierce/Thermo) and equal amounts of total protein (25–40 µg) per lane were resolved in a 4–12% gradient Bis-Tris gels (Novex, Invitrogen) under reducing conditions. After transfer to PVDF membranes (Amersham Biosciences/GE Healthcare), the filters were probed with the following antibodies: A8717 (Sigma; APP C-terminus), dNGluc (Proteintech Group Inc) and GAPDH (Millipore). After incubation with horseradish-conjugated secondary antibodies, the signal was developed using ECL Western blotting detection reagent (Pierce/Thermo).
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5

Monocyte Culture and Seeding

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Purified monocytes were suspended in RPMI‐1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mm L‐glutamine, 100 U mL−1 penicillin, and 100 µg mL−1 streptomycin (all from Sigma‐Aldrich) (henceforth referred to as “complete medium”) and seeded at three million cells per well in 6‐well polystyrene plates (Corning Life Sciences).
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6

FESEM Analysis of Mono- and Dual-Species Biofilms

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For FESEM analysis, the mature mono- and dual-species biofilms were established on glass coverslip in 6-well polystyrene plates (Corning, NY, USA) covered, as described above. Following biofilm formation, the harvested mono- and dual-species biofilms were washed three times with PBS and then fixed with 2.5% glutaraldehyde for 6 h at 4 °C. Subsequently, the samples were gradually dehydrated in graded ethanol (25, 50, 75, 90, 95, and 100%) for 10 min and then air-dried, gold-coated, and examined under a FESEM (Quanta FEG 650, FEI, Hillsboro, OR, USA).
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7

Biofilm Extraction and Metabolite Analysis

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The mature mono- and dual-species biofilms were established on 6-well polystyrene plates (Corning, NY, USA) as described above. After 21 h of incubation, the planktonic cells were removed with sterilized PBS. The biofilms were incubated in sterile saline solution for another 3 h at room temperature. The mixtures were subsequently sonicated and then centrifuged for 3 min at 4 °C to harvest the biofilm samples [12 (link)]. The supernatants were mixed with 1 mL of pre-cooled methanol/acetonitrile/water solution (2:2:1, v/v) successively for 1 min of vortexing, and then the mixtures were sonicated at low temperature for 30 min. Subsequently, the samples were allowed to stand at –20 °C for 60 min, followed by centrifugation at 14,000× g for 20 min at 4 °C. The supernatant was collected and vacuum freeze-dried for sample analysis. Finally, dried samples were dissolved in 100 µL acetonitrile/water (1:1, v/v), followed by vortexing and ultrasonic treatments. Next, each sample was centrifuged at 14,000× g at 4 °C for 15 min, and then each sample was transferred to a sample bottle with a lining tube for subsequent chemical analysis.
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8

Transfection of HEK293 Cells with pEGFP-mTRPM7

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HEK293 cells were chemically transfected with pEGFP-mTRPM7 and pEGFP-mTRPM7 S1107R plasmids using polyethyleneimine (PEI) (Polysciences, Warrington, PA) or LT1 (Mirus Bio, Madison, WI) by mixing with DNA and Dulbecco’s phosphate-buffered saline (DPBS) and adding to cells in 6-well polystyrene plates (Corning Incorporated, Kennebunk, ME) (Zhelay et al., 2018 (link)). Plasmid DNA was purified using PureYield plasmid midiprep kit (Promega, Madison, WI).
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9

HeLa Cell Plaque Assay for Sf301

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This was carried out according to Oaks, Wingfield & Formal (link) (1985 (link)) using confluent HeLa cell monolayers. Briefly, HeLa cells to be used in the plaque assay were grown to 100% confluency in 6-well polystyrene plates (Corning) in DMEM supplemented with 10% FBS. One hour before the infection, cell culture medium were changed into fresh medium and Sf301 or its LPS mutants from mid-exponential phase (m.o.i 100:1) was added to the cells and subsequently incubated at 37°C for 90 min. During this adsorption or attachment phase, the plates were rocked every 30 min to assure uniform distribution of the plaque-forming bacteria. Next, an agarose overlay (5 ml) consisting of DMEM, 5%FBS, 25 µg of gentamicin per ml, and 0.5% agarose was added to each plate. The plates were incubated at 37°C in a humidified 5% CO2 and examined daily for up to 3 days for plaque formation. The agar was carefully removed three days later and the HeLa monolayers were stained by Giemsa staining kit (Yeasen, Shanghai, China) in order to visualize the plaques.
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