Static oral multi-species biofilms were developed in 24-well glass-bottom Sensoplate microplates (Greiner Bio-One, Monroe, NC) using CFS as the sole nutrient source and CCS as the inoculum. When required, L-arginine HCl (LAHCl) was added to CFS at final concentrations between 50 μM–500 mM (10-fold increments). 1.5 mL of CFS (negative control) and supplemented CFS of different LAHCl concentrations were added to each well. Wells were inoculated with 15 μL of CCS. After incubation at 37°C for 22 h, wells were treated with BacLight LIVE/DEAD viability stain (3.34 μM Syto 9 and 20 μM propidium iodide) (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. After 30 min, the stained biofilms were washed with 1 mL of phosphate-buffered saline (PBS; pH: 7.4) three times, and examined microscopically.
Sensoplate microplate
Manufactured by Greiner
SensoPlate microplates are multi-well plates designed for various laboratory applications. They provide a standardized platform for performing assays, experiments, and sample analysis. The core function of SensoPlate microplates is to facilitate the organized and efficient handling of multiple samples or reactions simultaneously in a compact format.
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Lab products found in correlation
Baclight live dead viability stain, by Thermo Fisher Scientific (1 mentions)
Synergy h1, by Agilent Technologies (1 mentions)
Lysotracker deep red, by Thermo Fisher Scientific (1 mentions)
Lysotracker green dnd 26, by Thermo Fisher Scientific (1 mentions)
Concanavalin a (cona), by Merck Group (1 mentions)
Plan apochromat 63 1.4 na, by Zeiss (1 mentions)
Coolsnap hq2 camera, by Olympus (1 mentions)
Delta vision inverted microscope, by Olympus (1 mentions)
Spinning disk confocal microscope, by Zeiss (1 mentions)
Zen 2 blue software, by Zeiss (1 mentions)
Axiocam 506 mono, by Zeiss (1 mentions)
5 protocols using sensoplate microplate
1
Developing Multispecies Biofilms with L-Arginine
2
Microplate-based Bacterial Growth Assay
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Attenuance (D600 nm) measurements were taken every 10 min from selective or non-selective NM minimal media subcultures grown with IPTG (100 µM) and continuous shaking for 18 h at 37 °C in a Synergy H1 microplate reader (BioTek). For subculturing, 96-well SensoPlate microplates with a clear, flat bottom (Greiner Bio-One) were used. Each 200 µl subculture was generated by dilution (1:100) from independent post-exponential rich media cultures; these 1 mL rich cultures were inoculated and grown in 96-well assay blocks as described above for GFP fluorescence assays.
3
Imaging of Adherent and Suspension Cells
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Cells were imaged in glass‐bottomed 96‐well SensoPlate microplates (Greiner Bio‐one Ltd). When staining HeLa cells, 1 × 104 cells were added per well and incubated for 2 days. When staining Jurkat cells, 1.5 × 105 cells/well were incubated on ice in 3.7% formaldehyde 20 mM HEPES in PBS for 10 min, added to poly‐l ‐lysine coated wells and centrifuged (350 g, 5 min, 4°C) to adhere them to the glass‐bottomed wells.
4
Visualizing Lysosomal Dynamics in Drosophila S2 Cells
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Drosophila S2 cells were plated for 3 h in 96-well plates (Sensoplate microplate; Greiner Bio-One) previously coated with Concanavalin A (0.5 μg/μl; C2010; Sigma-Aldrich). Then cells were incubated for 1 h with LysoTracker Deep Red (L12492; Invitrogen) or LysoTracker Green DND-26 (L7526; Invitrogen) at 75 nM. LysoTracker was washed with fresh medium, and images were taken with a confocal microscope LSM880 with non-linear optics using a 63×/1.4 Plan-Apochromat, DIC objective. Images were treated using ZEN lite software, ImageJ software, and Photoshop.
5
Live-cell imaging of moesin-GFP and Slik-GFP
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The stable cell line expressing moesin-GFP in combination with α-tubulin-mCherry has been described previously (Roubinet et al., 2011 (link)). Stable cell lines expressing SlikWT/NCD*/CTD*-GFP (under pMt promoter) were generated upon Gibco Hygromycin B (Thermo Fisher Scientific) selection. SlikWT/NCD*/CTD*-GFP expression was induced with copper sulfate (CuSO4; 0.7mM), and cells were analyzed 36 h after induction. For time-lapse microscopy, 50 × 103 cells were plated in 96-well glass-bottom plates (82050-792, SensoPlate microplate; Greiner Bio-One) and filmed either overnight (dsRNA treatments) or for 1 h (drug treatments) in an environmental chamber at 27°C in Schneider’s medium. Cells expressing moesin-GFP were imaged using a DeltaVision inverted microscope (Olympus) with a Plan Apochromat 60×/1.42 NA oil lens (equipped with a CoolSNAP HQ2 camera) and controlled by SoftWoRx software. Cells expressing Slik-GFP were imaged using a Carl Zeiss confocal spinning disk microscope with a Plan Apochromat 63×/1.4 NA oil differential interference contrast lens (equipped with a Zeiss Axiocam 506 mono camera) and controlled using ZEN 2 blue software (Carl Zeiss Microscopy). Representative images were prepared for publication using SoftWoRx, ImageJ (National Institutes of Health), and Photoshop (Adobe) software.
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