The following materials were purchased from the manufacturers indicated: heparin, heparan sulfate, chondroitin sulfate A, B and C, heparinases I and III, chondroitinase ABC, fluorescein isothiocyanate (FITC), GenElute PCR clean-up kit, phospholipase C phosphatidylinositol-specific (PI-PLC) from Bacillus cereus, all from Sigma-Aldrich (St. Louis, MO, USA); 2-O, 6-O and N-desulfated heparins from Amsbio (Abingdon, UK); Dulbecco’s Modified Eagle’s minimal essential medium (DMEM) and Minimum Essential Medium (MEM), fetal bovine serum, penicillin-streptomycin, and PBS-phosphate-buffered saline from Gibco-Thermo Fischer Scientific (Waltham, MA, USA); Brain-Heart Infusion broth from Pronadisa (Madrid, Spain); RNeasy Kit and RNase-Free DNase from Qiagen (Hilden, Germany); High-Capacity cDNA Reverse Transcription Kit and PowerSYBR Green PCR Master Mix from Applied Biosystems (Foster City, CA, USA). Synthetic peptides were from Abyntek Biopharma (Derio, Spain); mouse monoclonal anti-syndecan 1 (CD138) from DakoCytomation (Carpinteria, CA, USA); and rabbit anti-syndecan 2, goat anti-syndecan 3 and rabbit anti-syndecan 4 polyclonal antibodies from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals were obtained from commercial sources and were of analytical grade.
Pbs phosphate buffered saline
Manufactured by Thermo Fisher Scientific
Sourced in United States
PBS (phosphate-buffered saline) is a commonly used buffer solution in laboratories. Its core function is to maintain the physiological pH and osmolarity of aqueous solutions, making it suitable for various applications such as cell culture, immunoassays, and protein purification.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Heparan sulfate, by Merck Group (1 mentions)
Heparin, by Merck Group (1 mentions)
Genelute pcr clean up kit, by Merck Group (1 mentions)
Chondroitinase abc, by Merck Group (1 mentions)
Heparinases 1 and 3, by Merck Group (1 mentions)
Phospholipase c phosphatidylinositol specific pi plc from bacillus cereus, by Merck Group (1 mentions)
Rneasy kit and rnase free dnase, by Qiagen (1 mentions)
Mouse monoclonal anti syndecan 1 cd138, by Agilent Technologies (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Fluorescein isothiocyanate (fitc), by Merck Group (1 mentions)
Phospho sapk jnk, by Cell Signaling Technology (1 mentions)
Ripa lysis and extraction buffer, by Merck Group (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Sapk jnk, by Cell Signaling Technology (1 mentions)
Phospho p38, by Cell Signaling Technology (1 mentions)
Caspase 8, by Novus Biologicals (1 mentions)
Phospho erk1 2, by Cell Signaling Technology (1 mentions)
Tnf α, by Cell Signaling Technology (1 mentions)
6 protocols using pbs phosphate buffered saline
1
Glycosaminoglycans Characterization Protocol
2
Molecular Mechanisms of UA-Induced Apoptosis
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UA (Santa Cruz, CA, USA) was over 98% purity, and its chemical structure is presented in Figure 1 . MTT (3-[4,5-dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide) was purchased from Merck (Darmstadt, Germany). BSA (Bovin Serum Albumin), TritonTM X-100, Paraformaldehyde, Trypan blue solution, and β-actin were purchased from Sigma-Aldrich (St. Louis, MO, USA). FBS (Fetal bovine serum), DMEM (Dulbecco’s Modified Eagle Medium), penicillin-streptomycin, and PBS (phosphate-buffered saline) were purchased from Gibco BRL (Grand Island, NY, USA); and DAPI (4′6-diamidino-2-phenylindole) was purchased from Invitrogen (Carlsbad, CA, USA). Pierce™ BCA Protein Kit was purchased from Thermo Fisher (Waltham, MA, USA). Western BrightTM Quantum HRP substrate solution was purchased from Advansta (Menlo Park, CA, USA). Tween-20 was purchased from Amresco (St. Louis, MO, USA); and RIPA lysis and extraction buffer was purchased from Millipore (Billerica, MA, USA). Antibodies to Fas, Bcl-2, and caspase-8 were purchased from Novus Biologicals (Littleton, CO, USA). Antibodies to TNF-α, FADD, Bax, Bcl-xL, Mcl-1, TCTP, caspase-3, PARP, PI3K, phospho-Akt, Akt, phospho-ERK1/2, ERK1/2, phospho-p38, p38, phospho-SAPK/JNK, and SAPK/JNK were purchased from Cell Signaling (Beverly, MA, USA).
3
Evaluating Apoptosis in NP Cells
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Human NP cells, NP Cell Growth Supplement and NP cell medium (NPCM) were purchased from ScienCell Research Laboratories, Inc. (Carlsbad, CA, USA), and Dulbecco's modified Eagle's medium (DMEM)/F-12 and fetal bovine serum (FBS) were obtained from HyClone; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Trypsin, Cell Counting kit-8 (CCK-8) and 17β-E2 were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). PBS (phosphate buffered saline) was obtained from Gibco; Thermo Fisher Scientific, Inc. The Annexin V-Fluorescein Isothiocyanate (FITC)/Propidium Iodide (PI) kit was purchased from BD Biosciences (Franklin Lakes, NJ, USA), and the caspase-3 activity kit, Hoechst staining kit and cell lysis buffer for western and immunoprecipitation were obtained from Beyotime Institute of Biotechnology (Shanghai, China). Z-DEVD-FMK was purchased from MedChem Express (Monmouth Junction, NJ, USA), ICI 182,780 was obtained from Sigma-Aldrich, Merck KGaA; and TNF-α was purchased from PeproTech, Inc. (Rocky Hill, NJ, USA).
4
Isolation and Cultivation of Glioblastoma Cells
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Cells were obtained from human glioblastoma tissues after tumor resection. Cell culture G01 was chosen for this research as it exhibits high expression of the CD133 cell surface marker. This culture was obtained by washing cells in Versen solution (Paneco, Russia), incubation with 0.25% trypsin-EDTA (Ethylenediaminetetraacetic acid) solution (Gibco, UK) at 37°C for 40 min, and disaggregation from tissue followed by centrifugation at 1,000 rpm for 5 min. Cells were cultivated in DMEM/F12 (Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12) medium (Gibco, UK) containing 1% L-glutamine (Paneco, Russia), 10% FBS (Fetal Bovine Serum) (Thermo Scientific, USA), and 1% HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (Sigma-Aldrich, USA) at 37°C with a humidified atmosphere of 5% CO2. Passaging was performed at 80% confluency, for which cells were washed in PBS (Phosphate Buffered Saline) (Gibco, UK) and incubated with a 0.25% trypsin-EDTA solution. After trypsin inactivation by fresh medium, cells were centrifuged at 1,000 rpm for 5 min, the supernatant was removed, fresh medium was added, and the cells were resuspended and plated into cultural flasks. The cell count was defined using Trypan Blue 1:1.
5
Vitreal Cell ROS Quantification
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Vitreous samples were quickly delivered to the laboratory and after a 1 : 2 dilution in Hank's Balanced Sodium Salt (HBSS), vitreous samples were placed on special 8-well slides (Nunc™ Lab-Tek II™ 8 wells; Thermo Scientific™) to let the adherence of vitreal cells to the slides (37°C for 30 min with 5% CO2). After gentle vitreous aspiration, adherent cells were exposed to a cell permeant reagent 2′,7′–dichlorofluorescin diacetate (H2DCFDA) working solution, according to the manufacturers' instructions (ab113851; DCFDA/H2DCFDA-Cellular ROS Assay Kit; Abcam, Cambridge, UK). Washed cells were thereafter counterstained with DAPI prepared in Phosphate Buffered Saline (PBS, 10 mM PB and 137 mM NaCl; pH 7.5; Invitrogen-Molecular Probes, Eugene, Oregon). Fluorescent cells (Ch1/green) having blue nuclei (Ch3/blue) were observed at the inverted Eclipse TE2000U microscope, and images were acquired by C1 software (Nikon, Tokyo, Japan). Channel series were carried out to reduce autofluorescence. Digital images (pixel size: 1024 × 1024 dpi) were converted into 8-bit TIFF images and subjected to densitometric analysis (ImageJ v1.43; http://rsb.info.nih.gov/ij/ ). Single integrated optical density (IntDen) was registered for fluorescent ROS expression at different stages (n = 5, optic fields/slide; ×40/dry 0.75 DIC M/N2), mean values ± SD were used for statistical analysis.
6
MTT Assay for HEK293 Cell Viability
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The human embryonic kidney cell line (HEK293) was purchased from the Korean Cell Line Bank for Biological Sciences (KCLB, Seoul, Korea). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Sigma (St. Louis, MO). Roswell Park Memorial Institute (RPMI) 1640 medium, phosphate-buffered saline (PBS), fetal bovine serum, penicillin/streptomycin, and trypan blue were purchased from Invitrogen Molecular Probes (Carlsbad, CA). The proliferation of HEK293 cells was measured using a colorimetric MTT assay. HEK293 cells were seeded onto a 96-well plate at a density of 1.0×104 cells/well. Briefly, the cells were incubated with different concentrations of samples for 24 h, followed by the addition of 20 µL MTT solution to each well at a final concentration of 0.5 mg/mL in PBS (pH 7.4). The plates were incubated for an additional 4 h at 37℃ in the dark. Following removal of the medium, the dye crystals were dissolved in 100 µL dimethyl sulfoxide, and the absorbance of each well was measured at 570 nm using a microplate reader (Infinite 200 pro, TECAN Ltd., Mannedorf, Switzerland). The cell viability was expressed as a percentage of the ratio of absorbance between treated and untreated groups.
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