A semi-static biofilm model was used to assess biofilm formation of S1 and S2, as described before[43 (link)]. Overnight bacterial cultures were diluted to an OD660 of 0.01 in 6 ml LB medium with 1%(wt/vol) glucose and added to a coverslip coated with poly-L-lysine (0.45 μm; diameter, 12 mm; Becton Dickinson) inside a well from a six-well polystyrene plate (Corning Inc.). Biofilms were grown at 30°C for 48 hat 120 rpm. After 48 h, the coverslips were washed with 0.85% (wt/vol) NaCl and the biofilms were chemically fixed with 8% (vol/vol) glutaraldehyde (Merck) for 20 min. Subsequently, the biofilms were stained with 15 μg/ml propidium iodide (PI) in 0.85% (wt/vol) NaCl that was removed after 15 min. The coverslips were transferred to glass microscope slides and analyzed by a confocal laser scanning microscope (CLSM) (Leica SP5), equipped with an oil plan-Neofluar ×63/1.4 objective. PI was excited at 488 nm. Z stacks were taken with an interval of 0.42 μm. Pictures were analyzed with LAS AF software (Leica), and biofilm thickness and biomass were quantified using Comstat[44 (link)]/Matlab R2013b software (the MathWorks). The average thickness and biomass of the biofilms were measured at ten randomly chosen positions and statistical significance determined by unpaired two-tailed Student's t-test. Experiments were performed twice in duplicate.
Poly l lysine (pll)
Manufactured by BD
Sourced in United States
Poly-L-lysine is a synthetic polymer composed of the amino acid lysine. It is commonly used as a surface coating for various laboratory equipment and materials to enhance cell attachment and adhesion.
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Lab products found in correlation
Collagen 1, by BD (2 mentions)
Fibronectin, by BD (2 mentions)
Las af software, by Leica (1 mentions)
Six well polystyrene plate, by Corning (1 mentions)
Matlab r2013b software, by MathWorks (1 mentions)
Glutaraldehyde, by Merck Group (1 mentions)
Elisa plate reader, by Agilent Technologies (1 mentions)
Countess automated counter, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated gelatin, by Thermo Fisher Scientific (1 mentions)
Fluoview software, by Olympus (1 mentions)
Fv500 confocal laser scanning microscope, by Olympus (1 mentions)
Collagen 4, by BD (1 mentions)
Ix70 confocal laser scanning microscope, by Olympus (1 mentions)
Fluoview fv1000, by Olympus (1 mentions)
Fluorescent microscope, by Nikon (1 mentions)
Triton x, by Merck Group (1 mentions)
Cardiac troponin t ctnt, by Abcam (1 mentions)
5 azacytidine, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
13 protocols using poly l lysine (pll)
1
Assessing Biofilm Formation Dynamics
2
BrdU-ELISA for DNA Proliferation
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BrdU-incorporated DNA was attached on a 96-well plate coated with poly-L-lysine (Becton Dickinson) and bound with POD-conjugated anti-BrdU antibody (Roche) for 30 min. After washing unbound antibodies with PBS, bound POD was activated by ultra-TMB-ELISA solution (Thermo Scientific), and the reaction was stopped after 5 min with 2 M H2SO4. The colorimetric signal was measured at a 450-nm wavelength in an ELISA plate reader (BioTek). The reading from a blank well was subtracted from all measurements, and error bars represent the standard deviation from three technical replicates. The result was confirmed by two independent measurements.
3
Dissociation and Culture of Pituitary Cells
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After sacrificing the mice, the anterior pituitaries were dissected, washed twice in HBSS, minced to 2-3 mm slivers, and dispersed to individual cells by incubating tissues under gentle agitation with 0.25% Trypsin type II-S, 0,5% Collagenase, and DNAase 1 µM in Neurobasal-A complete medium for 45 minutes at 37°C. Freshly dissociated cells were centrifuged (1000 RPM, at RT for 5 min), and resuspended in 1 ml of the complete medium for the cell count (Countess automated counter, Invitrogen, Carlsbad, CA, USA) and appropriate seeding.
For RT-qPCR, Western Blot and immunoprecipitation analysis, the pituitary cells were seeded in 35 mm cell culture dishes coated with poly-L-Lysine at a density of 5x10 5 cells/ml. For the caspase activity assays, we seeded cells in 96-well tissue culture plates coated with poly-L-Lysine at a density of 5x10 3 cells/well in 100 μl of the complete medium. For immunofluorescence, cells were seeded in 8-chamber cell culture slides coated with poly-L-Lysine (BD Biosciences, San Jose, CA, USA), at a density of 2x10 5 cells/slide in a 250 μl volume of complete culture medium.
For RT-qPCR, Western Blot and immunoprecipitation analysis, the pituitary cells were seeded in 35 mm cell culture dishes coated with poly-L-Lysine at a density of 5x10 5 cells/ml. For the caspase activity assays, we seeded cells in 96-well tissue culture plates coated with poly-L-Lysine at a density of 5x10 3 cells/well in 100 μl of the complete medium. For immunofluorescence, cells were seeded in 8-chamber cell culture slides coated with poly-L-Lysine (BD Biosciences, San Jose, CA, USA), at a density of 2x10 5 cells/slide in a 250 μl volume of complete culture medium.
4
Culturing Primary Cortical Neurons
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Primary cultured neurons were used as a model reported to develop morphological (Cao et al. 2015 (link)) as well as functional changes that recapitulate several hallmarks of neurodevelopment observed in vivo (Feller 1999 (link); Stafford et al. 2009 (link)). Procedures culturing cortical neurons from postnatal day 0 or 1 (both male and female) C57BL/6J WT and knock-in mice were performed as described previously (Cao et al. 2011b (link)). In brief, the dissociated cells were suspended in prewarmed neurobasal complete (NBC) medium [neurobasal medium supplemented with GS21 (2%, v/v), GlutaMAX (1%, v/v), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; )] containing 5% fetal bovine serum (FBS). Suspended cells were plated onto poly L-lysine ( in borate buffer) precoated 96-well imaging plate (BD Biosciences) at densities of 2,000; 75,000; and 130,000 cells/well for axonal and dendritic outgrowth, SCO, and microelectrode array (MEA) measurements, respectively. A final concentration of of cytosine dissolved in FBS-free NBC was added at 24–30 h after plating. The neurons were maintained at 37°C with 5% and 95% humidity until use at specified DIV.
5
Visualizing Extracellular Matrix Degradation
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The experiments were done as previously described.21 Briefly, sterile coverslips coated with poly‐l ‐lysine (BD Biosciences) were washed with PBS and incubated with 0.5% glutaraldehyde for 15 minutes at room temperature. Coverslips were washed with PBS and coated with FITC‐conjugated gelatin (Invitrogen) for 10 minutes. After washing with PBS, coverslips were incubated with sodium borohydride for 1 minute and washed with PBS. Cells were seeded in 24‐well plates containing prepared coverslips coated with fluorescent gelatin and incubated at 37°C for 12 hours. Next, cells were fixed with 4% formaldehyde and labelled for filamentous actin with Alexa Fluor 568‐phalloidin. Images were taken using the Olympus FV500 confocal laser scanning microscope and FluoView software (Olympus). Sites of degraded matrix were visible as dark areas (spots) in the bright green fluorescent gelatin matrix. The area of gelatin digestion was calculated for 40 cells per condition using ImageJ software.20
6
Cell Adhesion Assay with Extracellular Matrix
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Ninety-six-microwell plates were coated overnight at 4°C with fibronectin, collagen IV, collagen I or poly-L-Lysine (10 μg/ml; BD Biosciences,). The cells were detached after over-night serum starvation and seeded on wells previously blocked with 1% bovine serum albumin at 6 × 104 cells/well density for 30min at 37°C. Additional experiments were accomplished after a pre-incubation of 30min with saturating concentrations of function-blocking anti-β1-integrin, control antibodies, pharmacological inhibitors, in presence or absence of 1mM Mn2+. After staining with DAPI, images of the total well surface area were acquired using Zeiss Axiovert 200 microscope and a Coolsnap HQ2 camera with a 10x objective. The total number of adherent cells per well was counted using NIH Image J software.
7
Quantifying Extracellular Matrix Proteolysis
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The experiment was performed using a previously described protocol [30 (link)]. Sterile coverslips coated with poly-l -lysine (BD Biosciences) were washed with PBS and incubated with 0.5% glutaraldehyde for 15 min in room temperature. Coverslips were washed with PBS and coated with FITC-conjugated gelatine (Invitrogen) for 10 min. After washing with PBS, coverslips were incubated with sodium borohydride for 1 min and washed again with PBS. To determine the proteolytic activity, cells were seeded onto prepared coverslips (5 × 104 cells) and grown with apelin peptides (100 nM) or ML221 (100 nM) for 24 h. Cells were fixed with 4% formaldehyde and stained with Phalloidin Alexa Fluor® 568 for filamentous actin detection. Images were collected using an Olympus IX70 confocal laser scanning microscope (Olympus, Tokyo, Japan) with FLUOVIEW FV1000 software (Olympus, Tokio, Japan). Images were analysed through quantification of darker areas lacking fluorescence, which reflected increased activity of ECM degrading proteases using ImageJ software [28 (link)]. The results were calculated as a percent of cells digesting gelatine and then presented as a percent of control. The experiments were performed three times.
8
Cardiomyogenic Differentiation of Cells
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The cells were grown on poly-L-lysine coated cover slips in 12 well tissue culture plates (BD Biosciences), and then treated with complete culture medium containing 10μM 5-azacytidine (Sigma-Aldrich, Merck’s Life Science, Germany). Control cells were treated with complete medium alone. After incubating for 24 h, the experimental and control cells were washed twice with PBS (Gibco) and further incubated in complete culture medium. The medium was changed every three days and the experiment was terminated at 21 days after 5-azacytidine treatment. Cells were fixed in 4% paraformaldehyde (Sigma-Aldrich), permeabilized with 0.1% triton X (Sigma-Aldrich), non-specific binding sites were blocked with 5% sheep serum (Hyclone) and 0.1% triton X in PBS. Thereafter, primary antibodies against beta myosin heavy chain (bMHC) (1:100 dilution), cardiac troponin T (cTnT) (1:100 dilution) (all from Abcam, UK) and desmin (AbD Serotec) were added. Hoechst 2.5mg/μl (Sigma-Aldrich) in PBS was added to stain nucleus. The cells were observed under fluorescent microscope (Nikon, Japan).
9
Quantifying Androgen Receptor Signaling in VCaP Cells
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For DHT induction experiments, VCaP cells were plated in 6-well plates coated with poly-L-lysine (BD BioCoat) in charcoal-treated FBS containing media at a density of 31k/cm2 (3x105 cells per well). The cells were allowed to adhere for 24 h and then dosed with 0.1% DMSO with or without polyamide 1 for 72 h followed by the addition of 0.01% ethanol in PBS with or without DHT (1 nM final concentration). Cells were harvested after additional 24 h incubation. Cells treated with etoposide and camptothecin (Sigma) were co-treated with DHT (1 nM) and harvested after a 16 h incubation. For native expression experiments, VCaP cells were plated as above but using standard FBS media and harvested after 72 h of treatment. For all experiments, the mRNA was extracted using the QIAGEN® RNeasy mini kit following the standard purification protocol. Samples were submitted to DNAse treatment using the TURBO DNA-free™ Kit (Ambion), and the mRNA was reverse-transcribed by using the Transcriptor First Strand cDNA Synthesis Kit (Roche). Quantitative PCR was performed by using the FastStart Universal SYBR Green Master (Rox) (Roche) on an ABI 7300 Real Time PCR System. Gene expression was normalized against GUSB. Primers used are referenced in S3 Fig .
10
VCaP Cell Viability Assay
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VCaP cells were plated at 2x104 per well in 96-well plates coated with poly-L-lysine (BD BioCoat). After 24 h, an additional volume of medium containing vehicle or polyamide was added to each well. All medium was removed following polyamide incubation at the indicated time points and replaced with one volume of WST-1 reagent (Roche) in medium according to manufacturer protocol. After 4 h of incubation at 37°C, the absorbance was measured on a FlexStation3 plate reader (Molecule Devices). The value of A(450 nm)-A(630 nm) of treated cells was referenced to vehicle treated cells. Non-linear regression analysis (Prism software, Graphpad) was performed to determine IC50 values.
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