Dulbecco s phosphate buffer saline

Manufactured by Thermo Fisher Scientific

Sourced in United States

Dulbecco's Phosphate Buffer Saline (DPBS) is a balanced salt solution commonly used in cell culture and biological research applications. It is designed to maintain the pH and osmotic balance of cells and tissues in various experimental settings. DPBS is a sterile, isotonic buffer solution that does not contain calcium or magnesium ions.

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Dulbecco’s phosphate buffered saline (DPBS) buffer Dulbecco’s phosphate buffer saline (DPBS) Dulbecco’s phosphate Phosphate buffer

24 protocols using dulbecco s phosphate buffer saline

Cytochrome P450 Membrane Fractions Purification

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Poly-l-lysine (PLL, M_w 30–70
kDa) and poly-l-ornithine (PLO, M_w 30–70 kDa) were purchased from Sigma-Aldrich (France). Polyelectrolyte
solutions were prepared in Dulbecco’s phosphate buffer saline
(PBS, pH 7.4, Thermo-Fisher, France) at a concentration of 1 mg/mL.
Membrane fractions (MF) overexpressing a human recombinant cytochrome
P450 (CYP) were obtained from Cypex (England). CYP1A2 was individually
coexpressed with human NADPH–cytochrome P450 reductase in E. coli following the procedure described elsewhere.^{44 (link)} Then, a cell fractionation method was used to
allow the purification of membrane fractions with highly enriched
recombinant enzymes. MF were provided in a storage buffer (50 mM Tris-acetate,
pH 7.6; 250 mM sucrose; 0.25 mM ethylenediaminetetraacetic acid) at
an estimated CYP concentration of 1 mM and a total protein concentration
of 10 mg/mL. Stock solutions were stored at −80 °C. MF
were diluted in PBS at pH 7.4 for the different experiments and used
immediately. All MF concentrations are given in estimated CYP concentration.

Quantin P., Colaço E., El Kirat K., Egles C., Ficheux H, & Landoulsi J. (2018). Layer-by-Layer Assembly of Nanosized Membrane Fractions for the Assessment of Cytochrome P450 Xenobiotic Metabolism. ACS Omega, 3(10), 12535-12544.

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Fibroblast Exposure to Urban Dust

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Human skin fibroblast (BJ) and lung fibroblast (WI-38) cells were obtained from American Tissue Type Collection (ATCC, Manassas, VA, USA). Eagle’s Minimum Essential Medium (EMEM), Dulbecco’s phosphate buffer saline (DPBS), fetal bovine serum (FBS), penicillin–streptomycin and gentamicin were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Ninety-six-well flat-bottom tissue culture plates were purchased from Corning (Corning, NY, USA). Millex 0.22 μm syringe filters were obtained from MilliporeSigma. Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Kumamoto, Japan). Dried urban road dust samples were UV sterilized overnight and suspended in ultrapure water (0.22 μm sterile filtered) at a concentration of 10 mg mL⁻¹. The sieved dust samples (< 100 μm) were briefly sonicated (5 min at 47 kHz, Branson 3,210) before application to cells.

Kim E.A, & Koh B. (2020). Utilization of road dust chemical profiles for source identification and human health impact assessment. Scientific Reports, 10, 14259.

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3D Organotypic Mucosal Model Development

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The 3D-OMM was developed based on the modification from the protocols developed by Chai et al. [26 (link)]. A mixture of 5 × 10⁵ HGFs and 5 × 10⁵ OKF6/TERT-2 was co-cultured onto the basement membrane side of the rehydrated acellular dermal membrane (Puros^® Dermis Allograft Tissue Matrix, Zimmer Biomet, Warsaw, IN, USA).
Prior to cell seeding, the membrane was cut into a round shape with a 12 mm diameter to fit into a 12 mm ring insert (Corning^® Costar^® Snapwell™ Insert, Corning Life Sciences, Corning, NY, USA). The membrane was rehydrated in a 5 mL Dulbecco’s phosphate buffer saline (Thermo Fisher Scientific Inc., Waltham, MA, USA) for 30 s and then immersed in a 5 mL DMEM for 15 min. The 3D-OMMs were fabricated inside 12 mm diameter inserts with a 0.4 µm pore size and a 1 × 10⁸ pore density/cm² in a six-well plate. The seeded membrane was submerged in 3 mL of K-SFM and incubated for five days. On the sixth day, the tissues were raised at the air-liquid interface (ALI) to promote epithelial differentiation. The cells were allowed to be stratified further for up to seven days in the incubator, with the media changed every two days. On day 12, the samples (3-YZP and IC) were placed on top of the models.

Nasarudin N.A., Razali M., Goh V., Chai W.L, & Muchtar A. (2023). Expression of Interleukin-1β and Histological Changes of the Three-Dimensional Oral Mucosal Model in Response to Yttria-Stabilized Nanozirconia. Materials, 16(5), 2027.

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Humanized Mouse Model of GVHD

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Experiments involving human blood and mice were approved by the respective Human and Animal Ethics Committees of the University of Wollongong (Wollongong, Australia). A humanised mouse model of GVHD was used as described [20] (link). Briefly, female NSG mice aged 6-8 weeks (Australian BioResources, Moss Vale, Australia) were injected i.p. daily (days -2 to day 11) with saline/0.2% DMSO (Sigma-Aldrich, St Louis, MO, USA) (vehicle) or vehicle containing CGS 21680 (Tocris Bioscience, Bristol, UK) (0.1 mg/kg). This injection schedule was based on that previously used for an A 2A agonist in an allogeneic mouse model of GVHD [13] . hPBMCs, isolated by density centrifugation using Ficoll-Paque PLUS (GE Healthcare; Uppsala, Sweden) and resuspended in Dulbecco's phosphate-buffer saline (ThermoFisher, Waltham, MA, USA), were injected i.p. (day 0) (10 x 10 6 hPBMCs/mouse). At 3 weeks post-hPBMC injection, mice were checked for engraftment by immunophenotyping of tail vein blood. Mice were monitored for signs of GVHD using a scoring system, giving a total clinical score out of 10, as described [21] (link). Mice were euthanized at 10 weeks post-injection of hPBMCs, or earlier if exhibiting a clinical score of ≥ 8 or a weight loss of ≥ 10%, according to the approved animal ethics protocol.

Geraghty N.J., Adhikary S.R., Watson D, & Sluyter R. (2019). The A_2A receptor agonist CGS 21680 has beneficial and adverse effects on disease development in a humanised mouse model of graft-versus-host disease. International immunopharmacology, 72.

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Porcine Cell Culture Reagents

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Dulbecco’s Modified Eagle’s Medium-Ham’s F12 (1:1, #31331, Gibco), Fetal Bovine Serum (#10500), 1 M HEPES (#15630), 7.5% Sodium bicarbonate (#25080), 10000 UI.ml⁻¹ Penicillin/Streptomycin (#15140), Dulbecco’s Phosphate Buffer Saline (#14190) were purchased from Gibco. Sodium selenite (S5261), CsA (#30024), Tac (F4679), insulin (I4011), triiodothyronine (T6397) and dexamethasone (D4902) were purchased from Sigma–Aldrich. Primary antibodies against porcine Cyclophilin A (ab41984, 1:1000) was purchased from Abcam, anti-β-Actin (MA1-91399, 1:10000), anti-Na+/K+ ATPase α subunit 1 (MA3-929, 1:2000), anti-Cofilin-1 (PA1-24931, 1:10000) and anti-Galectin-1 (#437400, 1:500) were purchased from ThermoFisher. Secondary antibodies were purchased from Sigma–Aldrich (Anti-Mouse IgG [whole molecule]-Peroxidase antibody produced in rabbit, A9044, 1:10,000; Anti-Rabbit IgG [whole molecule]-Peroxidase antibody produced in goat, A9169, 1:10,000) and ThermoFisher (F[ab’]2-Goat anti-Rabbit IgG [H+L] Secondary Antibody, HRP, A24531, 1:10000).

Burat B., Gonzalez J., Sauvage F.L., Aouad H., Arnion H., Pinault E., Marquet P, & Essig M. (2019). Sum of peak intensities outperforms peak area integration in iTRAQ protein expression measurement by LC-MS/MS using a TripleTOF 5600+ platform. Bioscience Reports, 39(6), BSR20190904.

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Evaluating Glioblastoma Cell Viability

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To assess cell viability, 96-well plates were coated with Matrigel under ice-cold conditions prior to plating with cells. Adherent glioblastoma cell lines were passaged and seeded at 4000 Cells/well in 96-well plates overnight, before treatment with a clinically relevant dose of TMZ (35 µM) [14 (link)] or RT (2 Gy). After 7 days, 50 µL of a 2 mg/mL solution of MTT Formazan (Sigma-Aldrich, USA) and Dulbecco’s Phosphate Buffer Saline (DPBS, without magnesium chloride and calcium chloride Gibco^TM, Waltham, MA, USA) was added to each well and incubated for 3 h. The medium/MTT solution was aspirated and DMSO (120 µL) added into each well. Each plate was shaken using an IKA^® MS 3 basic shaker (Sigma Aldrich, Saint Louis, MO, USA) at 600 rpm for 2 min. Absorbance was read at 570 nM using the SPECTROstar^®Nano microplate reader (BMG LABTECH, Ortenberg, Germany).

Lozinski M., Bowden N.A., Graves M.C., Fay M., Day B.W., Stringer B.W, & Tooney P.A. (2022). Transcriptomic Profiling of DNA Damage Response in Patient-Derived Glioblastoma Cells before and after Radiation and Temozolomide Treatment. Cells, 11(7), 1215.

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Hydrogel-Based 3D Cell Culture

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Poly (ethylene) glycol diacrylate 10,000 kDa (PEGDA) was procured from Laysan Bio Inc. (Alabama). Gelatin from porcine skin, photo-initiator (2-Hydroxy-4’-(2-hydroxyethoxy)-2-methylpropiophenone), methacrylic anhydride, ethylene glycol, and bovine serum albumin (BSA) were obtained from Sigma-Aldrich (St. Louis, MO). Geltrex™ LDEV-free reduced growth factor basement membrane, Dulbecco’s Phosphate Buffer Saline (DPBS), Dulbecco’s Modified Eagle Medium (DMEM), Penicillin-Streptomycin (Pen Strep), 0.25% Trypsin-EDTA, Fetal Bovine Serum (FBS), and collagenase Type I were acquired from Gibco by Life Technologies (Grand Island, NY). Hoechst 33342, Alexa Fluor™ 488 phalloidin, methanol, acetone, and Slide-A-Lyzer Dialysis Cassette G2 (10k MWCO) were obtained from Thermofisher Scientific (Waltham, MA). XTT Proliferation Assay kit was purchased from ATCC (Manassas, VA). Endothelial cell basal medium-2 (EBM-2) and EGM-2 SingleQuots growth factors were obtained from Lonza (Walkersville, MD).

Nasser M., Wu Y., Danaoui Y, & Ghosh G. (2019). Engineering Microenvironments Towards Harnessing Pro-angiogenic Potential of Mesenchymal Stem Cells. Materials science & engineering. C, Materials for biological applications, 102, 75-84.

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Visualizing Cell Spreading in Gelatin Hydrogels

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Cell spreading and microsphere distribution within the gelatin matrix were evaluated after 1 and 14 days of culture. Hydrogels were fixed in paraformaldehyde 4% (v/v) for 15 min at room temperature. Subsequently, samples were submerged in sucrose (Sigma-Aldrich) solution 30% (w/v) overnight, immersed in OCT (Tissue Tek) and stored at −80 °C. Hydrogel sections of 30 μm were obtained using a cryostat (Leica CM 1860 UV) and placed on SuperFrost slides (Thermo Scientific).
Samples were washed twice with Dulbecco’s Phosphate Buffer Saline ((DPBS) Gibco) and permeabilised using Triton X-100 (Sigma-Aldrich) 0.1% (v/v) in DPBS for 10 min. Permeabilisation solution was removed, and samples were washed twice with DPBS. Slides were incubated with Rhodamine Phalloidin (ActinRed 555 ReadyProbes Reagent, Invitrogen, Waltham, MA, USA), following the manufacturer’s instructions, and Hoechst 3342 (1:250) for 1 h. Slides were finally washed with DPBS, and a mounting medium was added. Representative images were taken using a fluorescence microscope (Nikon Eclipse 80i). Gelatin hydrogels without microspheres (Gel) were used as controls.
Non-cryosectioned hydrogels were also observed in a confocal microscope (Leica DMI8), following the same staining protocol, and image processing for 3D reconstructions was performed using ImageJ software.

Guillot-Ferriols M., García-Briega M.I., Tolosa L., Costa C.M., Lanceros-Méndez S., Gómez Ribelles J.L, & Gallego Ferrer G. (2022). Magnetically Activated Piezoelectric 3D Platform Based on Poly(Vinylidene) Fluoride Microspheres for Osteogenic Differentiation of Mesenchymal Stem Cells. Gels, 8(10), 680.

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Microfluidic Device Fabrication and Cell Culture

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SU-8 2050 and its developer were purchased from MicroChem Corp. (USA) and used for microfabrication of microfluidic device master mold on a silicon wafer obtained from Nano-BAZAR (Iran). SYLGARD^® 184 polydimethylsiloxane (PDMS) kit, and Tygon tubing were purchased from Dow Corning (USA). MCF7 and MDA-MD-231 breast cancer cell lines were purchased from Pasteur Institute (Tehran, Iran). Cell culture reagents including Dulbecco’s phosphate buffer saline (DPBS), Dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS), trypsin-Ethylenediaminetetraacetic acid (EDTA), penicillin/streptomycin, and Geltrex^® were purchased from Gibco (Thermofisher Scientific, USA) while CellTrackers were obtained from Invitrogen (Thermofisher Scientific, USA).

Samandari M., Rafiee L., Alipanah F., Sanati-Nezhad A, & Javanmard S.H. (2021). A simple, low cost and reusable microfluidic gradient strategy and its application in modeling cancer invasion. Scientific Reports, 11, 10310.

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Rat Tail Collagen Type I Protocol

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Rat tail high concentration collagen type I was obtained from Corning (Bedford, MA). Sodium hydroxide, fluorescein isothiocyanate-dextran of 150K MW (FITC-dextran), sodium acetate, cysteine hydrochloride, sodium phosphate monobasic, sodium phosphate dibasic, papain suspension, cytochalasin D, and (–)-blebbistatin were procured from Sigma Aldrich (St. Louis, MO). Y-27632 and Na₂EDTA were purchased from EMD Millipore (Kankakee, IL). Dulbecco’s Phosphate Buffer Saline (DPBS), RPMI Medium 1640, Penicillin-Streptomycin (Pen Strep), 0.25% Trypsin-EDTA, fetal bovine serum (FBS), and collagenase Type I were acquired from Gibco by Life Technologies (Grand Island, NY). D-(–)-Ribose, Hoechst 33342, and Alexa Fluor™ 488 phalloidin were obtained from Thermofisher Scientific (Waltham, MA). AlamarBlue™ cell viability reagent was purchased from Invitrogen by Thermo Fisher Scientific (Eugene, OR). Acetone, methanol, and glacial acetic acid were acquired from Fisher Chemical (New Jersey). Endothelial cell basal medium-2 (EBM-2) and EGM-2 SingleQuots growth factors were obtained from Lonza (Walkersville, MD).

Nasser M, & Ghosh G. (2022). Engineering tumor constructs to study matrix-dependent angiogenic signaling of breast cancer cells. Biotechnology progress, 38(3), e3250.

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