Plastic coverslip, by Thermo Fisher Scientific - Product details

1

Quantification of Metabolic Parameters

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Kits for Glucose, insulin, total cholesterol, triglycerides, HDLc and LDLc were purchased from Abbott Laboratories (Abbott Park, IL, USA). HsCRP kits were supplied by Beckman Corp (Brea, A, USA). The HbA1c kit was purchased from Menarini Diagnostics (Florence, Italy). The MPO kit was purchased from Cayman Chemical (Michigan, USA).
Glucose, trypan blue, arginine, glutathione reductase, H₂O₂, haemoglobin, RPMI1640 supplemented with 20 mM HEPES, HBSS, TNF-α, human serum albumin (HSA, Albuminate 25%) and fibronectin were obtained from Sigma-Aldrich (Sigma Chem. Co., St. Louis, MO, USA). Dextran was acquired from Fluka (St. Louis, MO, USA). HBSS was supplied by Cambrex (Verviers, Belgium). DCFH-DA and Mitosox tracker were provided by Calbiochem (San Diego, CA, USA). Dulbecco’s PBS—with (DPBS⁺) or without (DPBS^-) Ca²⁺ and Mg²⁺—endothelial cell growth medium culture media and fetal bovine serum were obtained from LONZA (Verviers, Belgium). Plastic coverslips (diameter of 25 mm) were purchased from Nunc (Thermo Fisher Scientific). PBS, collagenase, and trypsin-EDTA were obtained from Invitrogen (Eugene, OR, USA). Ficoll-Paque TM Plus was purchased from GE Healthcare (Little Chalfont, Buckinghamshire,UK).

Victor V.M., Rovira-Llopis S., Bañuls C., Diaz-Morales N., Martinez de Marañon A., Rios-Navarro C., Alvarez A., Gomez M., Rocha M, & Hernández-Mijares A. (2016). Insulin Resistance in PCOS Patients Enhances Oxidative Stress and Leukocyte Adhesion: Role of Myeloperoxidase. PLoS ONE, 11(3), e0151960.

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2

Quantifying Hypoxia-induced Actin Changes

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Cells were cultured on plastic coverslips (Nunc), transfected with scramble or ZIP12 siRNA and exposed to hypoxia as described previously. After 48h exposure, cells were fixed with 4% formaldehyde solution in phosphate buffered saline (PBS) for 10 minutes at room temperature. Cells were then incubated with Alexa 568-conjugated phalloidin (1/200; Invitrogen) for F-actin detection under confocal microscopy. Sequential XYZ-sections (approximately 12 section of 1μm² / view) were obtained and 3D images were reconstructed. Quantification of actin stress fibres was determined by volume rendering in Image-J. Actin volume per cell was expressed as fold increase from normoxic control (value set at 1).

Zhao L., Oliver E., Maratou K., Atanur S.S., Dubois O.D., Cotroneo E., Chen C.N., Wang L., Arce C., Chabosseau P.L., Ponsa-Cobas J., Frid M.G., Moyon B., Webster Z., Aldashev A., Ferrer J., Rutter G.A., Stenmark K.R., Aitman T.J, & Wilkins M.R. (2015). The zinc transporter, ZIP12, regulates the pulmonary vascular response to chronic hypoxia. Nature, 524(7565), 356-360.

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3

BDE-47 Exposure Effects on Neurotoxicity and Oxidative Stress

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BDE-47 (2, 2′, 4, 4′-tetrabromodiphenyl ether; 99.9%) was obtained from AccuStandard Inc. (New Haven, CT). Poly-D-lysine, glutathione ethylester (GSHEE), dimethylsulfoxide (DMSO), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO). Hoechst 33342-DNA binding dye was from Molecular Probes (Eugene, OR), while 2,7′-dichlorofluorescein diacetate, neurobasal-A medium, fetal bovine serum and gentamycin were from Invitrogen (Carlsbad, CA). Melatonin was from Tocris Cookson Inc. (Elisville, MO) and the protein bicinchoninic acid (BCA) assay was from Thermo Fischer Scientific (Rockford, IL). The ELISA kit for measurement of serum thyroxine (T4) and triiodothyronine (T3) was purchased from Alpha Diagnostic International (San Antonio, TX). The ELISA kit for measurement of BDE-47 was purchased from Abraxis (Warminster, PA). The Thiobarbituric Acid Reactive Substances (TBARS) assay kit and the enzyme immunoassay (EIA) kit for measuring 8-isoprostane were purchased from Cayman Chemical Company (Ann Arbor, MI). Glass coverslips were from Fischer Scientific (Federal Way, WA) and the plastic coverslips from Nunc (Rochester, NY).

Costa L.G., Pellacani C., Dao K., Kavanagh T.J, & Roque P.J. (2015). The brominated flame retardant BDE-47 causes oxidative stress and apoptotic cell death in vitro and in vivo in mice. Neurotoxicology, 48, 68-76.

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4

Patch Clamp Analysis of SPIO-Labeled BM-MSCs

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SPIO labeled BM-MSCs and SPIO labeled induced cells with neural-like morphology were chosen for whole-cell patch clamp recording. Plastic cover slips (Nunc) containing a monolayer cells were transferred to a recording chamber on the stage of an inverted microscope. The culture medium was replaced with extracellular solution containing: 140 mMNaCl, 5 mMKCl, 1 mM CaCl₂, 1 mM MgCl₂, 10 mM glucose, 10 mM HEPES (pH = 7.3); pipettes were filled with an intracellular-like solution containing 140 mMKCl, 5 mMNaCl, 1 mM CaCl₂, 10 mM HEPES, 5 mM EGTA, 2 mM Mg-ATP. The resistant of fire-polished pipettes was 5–10 MΩ. All experiments were performed at room temperature. Ionic currents were recorded using the patch-clamp whole-cell configuration with Axopatch-200Bamplifier (Axon Instruments, USA)and digitized using a digidata 1322A A/D converter. Data were analyzed using pClamp10.1 and Originpro 8.0 software.

Zhang R., Li J., Li J, & Xie J. (2014). Efficient In Vitro Labeling Rabbit Bone Marrow-Derived Mesenchymal Stem Cells with SPIO and Differentiating into Neural-Like Cells. Molecules and Cells, 37(9), 650-655.

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5

Quantifying Hypoxia-induced Actin Changes

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Cells were cultured on plastic coverslips (Nunc), transfected with scramble or ZIP12 siRNA and exposed to hypoxia as described previously. After 48h exposure, cells were fixed with 4% formaldehyde solution in phosphate buffered saline (PBS) for 10 minutes at room temperature. Cells were then incubated with Alexa 568-conjugated phalloidin (1/200; Invitrogen) for F-actin detection under confocal microscopy. Sequential XYZ-sections (approximately 12 section of 1μm² / view) were obtained and 3D images were reconstructed. Quantification of actin stress fibres was determined by volume rendering in Image-J. Actin volume per cell was expressed as fold increase from normoxic control (value set at 1).

Zhao L., Oliver E., Maratou K., Atanur S.S., Dubois O.D., Cotroneo E., Chen C.N., Wang L., Arce C., Chabosseau P.L., Ponsa-Cobas J., Frid M.G., Moyon B., Webster Z., Aldashev A., Ferrer J., Rutter G.A., Stenmark K.R., Aitman T.J, & Wilkins M.R. (2015). The zinc transporter, ZIP12, regulates the pulmonary vascular response to chronic hypoxia. Nature, 524(7565), 356-360.

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6

Electron Microscopy Sample Preparation

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Cells were differentiated on plastic coverslips (Thermo Fisher Scientific) or in the microfluidic organ-on-a-chip device by following protocols described above. Cells were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (Electron Microscopy Sciences) for 1 h followed by 1% osmium tetroxide in 0.1 M sodium cacodylate (Electron Microscopy Sciences) for 1 h and then dehydrated in ascending grades (30%, 50%, 70%, 80%, 90%, 95% and 100%) of ethanol. Samples were then chemically dried with hexamethydisilazane (Electron Microscopy Sciences) in a desiccator for overnight. Before imaging, samples were mounted and sputter-coated with a thin layer of gold (for coverslip samples) or 5 nm layer of platinum-palladium (for microfluidic devices) and imaged using a Zeiss Supra55VP field emission microscope with a secondary detector.

Musah S., Mammoto A., Ferrante T.C., Jeanty S.S., Hirano-Kobayashi M., Mammoto T., Roberts K., Chung S., Novak R., Ingram M., Fatanat-Didar T., Koshy S., Weaver J.C., Church G.M, & Ingber D.E. (2017). Mature induced-pluripotent-stem-cell-derived human podocytes reconstitute kidney glomerular-capillary-wall function on a chip. Nature biomedical engineering, 1, 0069.

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7

Electron Microscopy of Magnetic Nanoparticle Uptake

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U87MG cells were seeded in 24-well plate with plastic coverslips (Thermo Fisher Scientific) to ~10⁶ cells/well and incubated with MNPs (100 µg/mL). An NdFeB magnet was placed underneath the plate for 5 minutes to facilitate sedimentation. After incubation, the medium with MNPs was removed and the cells were gently washed with phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 12 mM phosphate). Incubation of a mixture of 3% (w/v) glutaraldehyde and 2% (w/v) PFA in 0.1 M cacodylate buffer (pH 7.4) at 4°C for 2 hours was used to fix the cells. The mixture was subsequently replaced by 1% osmium tetroxide for 1 hour. Cells were dehydrated in graded ethanol and embedded in Epon resin. Sections with 80 nm thickness were obtained, counterstained with 4% uranyl acetate in hydrogen peroxide for 2 hours and 0.4% lead citrate for 10 minutes, and were examined with TEM (H-7500; Hitachi, Tokyo, Japan) at Department of Anatomic Pathology, Chang Gung Memorial Hospital, Linkou.

Siow W.X., Chang Y.T., Babič M., Lu Y.C., Horák D, & Ma Y.H. (2018). Interaction of poly-l-lysine coating and heparan sulfate proteoglycan on magnetic nanoparticle uptake by tumor cells. International Journal of Nanomedicine, 13, 1693-1706.

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8

Electron Microscopy Sample Preparation

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Cells were differentiated on plastic coverslips (Thermo Fisher Scientific) or in the microfluidic organ-on-a-chip device by following protocols described above. Cells were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (Electron Microscopy Sciences) for 1 h followed by 1% osmium tetroxide in 0.1 M sodium cacodylate (Electron Microscopy Sciences) for 1 h and then dehydrated in ascending grades (30%, 50%, 70%, 80%, 90%, 95% and 100%) of ethanol. Samples were then chemically dried with hexamethydisilazane (Electron Microscopy Sciences) in a desiccator for overnight. Before imaging, samples were mounted and sputter-coated with a thin layer of gold (for coverslip samples) or 5 nm layer of platinum-palladium (for microfluidic devices) and imaged using a Zeiss Supra55VP field emission microscope with a secondary detector.

Musah S., Mammoto A., Ferrante T.C., Jeanty S.S., Hirano-Kobayashi M., Mammoto T., Roberts K., Chung S., Novak R., Ingram M., Fatanat-Didar T., Koshy S., Weaver J.C., Church G.M, & Ingber D.E. (2017). Mature induced-pluripotent-stem-cell-derived human podocytes reconstitute kidney glomerular-capillary-wall function on a chip. Nature biomedical engineering, 1, 0069.

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9

Astrocyte-Motor Neuron Co-culture Protocol

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APCs were plated at a density of 1.5x10⁵ cells per well in 24‐well plates on plastic coverslips (Thermo Scientific) coated with laminin (Sigma), fibronectin (Sigma), and Matrigel (BD Biosciences) in NSCR EF20 medium for 5–7 days followed by differentiation into astrocytes for a further 2 weeks in AstroMED CNTF medium. Control iPSC‐derived MN progenitors were dissociated with the Papain Dissociation System (Worthington Biochemical) and plated at a density of 5 × 10⁴ cells per well on top of the astrocytes once the astrocyte medium had been removed. MN plate down medium consisted of Neurobasal medium, 1% penicillin/streptomycin, 0.5% GlutaMAX, 0.5% B‐27, 0.5% N‐2 Supplement, 20 ng/ml basic FGF, 1 μM retinoic acid, 1 μM purmorphamine, 1 μM mouse Smo agonist SAG (Merck Millipore). Twenty‐four hours post MN plating, 20 ng/ml CNTF; R&D, 10 ng/ml GDNF, and 10 μM forskolin were added, with this medium used until day 14, feeding every 3 days. From day 14, RA, SAG, purmorphamine and forskolin was removed from the medium, with cells then maintained for up to 10 weeks.
Population cell viability of control iPSC‐derived MNs was performed with the observer blinded to the cell lines by counting the number of MNs on astrocytes stained with the MN marker SMI‐32, the apoptotic marker caspase‐3 and the nuclear marker DAPI. Twenty images were taken from each line at weeks 5–6 and 7–10 post‐plating.

Zhao C., Devlin A., Chouhan A.K., Selvaraj B.T., Stavrou M., Burr K., Brivio V., He X., Mehta A.R., Story D., Shaw C.E., Dando O., Hardingham G.E., Miles G.B, & Chandran S. (2019). Mutant C9orf72 human iPSC‐derived astrocytes cause non‐cell autonomous motor neuron pathophysiology. Glia, 68(5), 1046-1064.

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10

Immunocytochemical Analysis of NF-κB p65

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Plastic coverslips (Thermo Fisher Scientific) with growing PDL cells were incubated in the presence or absence of F. nucleatum for 90 min. Following the immunocytochemistry method as described above, the cells were fixed and permeabilized and then blocked with nonfat dry milk (Bio-Rad) for 1 h. The slides were subsequently incubated with a rabbit anti-nuclear factor-κB p65 (E498) primary antibody (Cell Signaling Technology, Danvers, MA, USA; 1 : 100) for 90 min at RT. After rinsing with PBS and incubating with CY3-conjugated goat anti-rabbit IgG secondary antibody (Abcam; 1 : 1000) for 45 min at RT, the expression of NF-κB p65 in cells was observed with the ZOE™ Fluorescent Cell Imager (Bio-Rad) with a 20x objective. The images were captured with an integrated digital 5MP CMOS camera. Untreated cells were used as a control.

Nokhbehsaim M., Damanaki A., Nogueira A.V., Eick S., Memmert S., Zhou X., Nanayakkara S., Götz W., Cirelli J.A., Jäger A, & Deschner J. (2017). Regulation of Ghrelin Receptor by Periodontal Bacteria In Vitro and In Vivo. Mediators of Inflammation, 2017, 4916971.

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Plastic coverslip

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22 protocols using plastic coverslip

Quantification of Metabolic Parameters

Quantifying Hypoxia-induced Actin Changes

BDE-47 Exposure Effects on Neurotoxicity and Oxidative Stress

Patch Clamp Analysis of SPIO-Labeled BM-MSCs

Quantifying Hypoxia-induced Actin Changes

Electron Microscopy Sample Preparation

Electron Microscopy of Magnetic Nanoparticle Uptake

Electron Microscopy Sample Preparation

Astrocyte-Motor Neuron Co-culture Protocol

Immunocytochemical Analysis of NF-κB p65

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