Female wistar rats were used in the following age groups: 3 months (equivalent to a young human adult aged 20 years), 12 months (equivalent to a middle-aged human aged 40 years) and 24 months (equivalent to an aged human greater than 80 years) (Collier and Coleman, 1991 (link)). The animals were individually housed in an environmentally controlled room under a 12 h alternating light/dark cycle at 23°C and were fed commercial rat chow and water ad libitum. All experiments were performed with the approval of the Animal Ethics Committee of the University of Sydney. Anesthesia was induced with a mixture of O2, NO2, and 5% halothane, followed by an intraperitoneal injection of sodium pentobarbitone (60 mg/kg), and perfused transcardially as previously described (Chan-Ling, 1997 (link); Mansour et al., 2008 (link)). Whole brain was removed, washed with phosphate buffered saline solution (Invitrogen) and used immediately for a variety of biochemical and histochemical procedures.
Phosphate buffered saline solution
Manufactured by Thermo Fisher Scientific
Sourced in United States, Spain, Japan, Canada, United Kingdom, Germany
Phosphate-buffered saline (PBS) solution is a commonly used buffer in biological research and clinical applications. It is a balanced salt solution that maintains a physiological pH and osmolarity, typically used to preserve the structure and function of cells, tissues, or biomolecules. PBS solution serves as a versatile medium for various laboratory procedures, such as cell culture, immunoassays, and sample preparation.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (3 mentions)
Reagents for reverse transcription, by Thermo Fisher Scientific (3 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
Lipopolysaccharide lps from escherichia coli 0111 b4 reference l2630, by Merck Group (2 mentions)
Penicillin and streptomycin media supplements, by Thermo Fisher Scientific (2 mentions)
Trypan blue, by Harvard Bioscience (2 mentions)
Dimethyl sulfoxide (dmso), by Avantor (2 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Glutamax 1, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by GE Healthcare (1 mentions)
Fungizone, by Thermo Fisher Scientific (1 mentions)
Ultrapure water, by Merck Group (1 mentions)
Human transferrin, by Thermo Fisher Scientific (1 mentions)
Trypsin edta 0.25, by Thermo Fisher Scientific (1 mentions)
Whatman, by Cytiva (1 mentions)
Gelatin, by Merck Group (1 mentions)
50 protocols using phosphate buffered saline solution
1
Aging-related Changes in Wistar Rats
2
Cytotoxicity Evaluation of Natural Spices
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Cinnamon, red chilli, turmeric and black pepper powders were purchased from the local grocery store. 0.22 µm cellulose ester mixed Whatman filter paper, and 29.3 mm diameter dialysis tube (MWCO: 3.5 kDa) were obtained from Fisher Scientific (Portugal). Dulbecco’s modified Eagle’s medium (DMEM) with high glucose content and all other chemicals were purchased from Sigma-Aldrich (Spain). Ultrapure water (18.2 MΩ·cm at 25 °C, Millipore USA) was used throughout the experiments. Dulbecco’s modified Eagle’s medium with nutrient mixture F-12 (DMEM/F-12) and GlutaMAX-I™, trypsin 0.25%–EDTA, antibiotic mixture of penicillin/streptomycin (10,000 U·mL−1/10,000 μg·mL−1), fungizone (250 μg·mL−1), human transferrin (4 mg·mL−1) and phosphate buffered saline solution (1× PBS) were obtained from GIBCO Invitrogen (Barcelona, Spain). Fetal bovine serum (FBS) was obtained from HyClone GE Healthcare (United Kingdom). HK-2 (ATTC® CRL-2190™) and LN-229 cells (ATTC® CRL-22611™) were obtained from ATCC (LGC Standards S.L.U., Spain).
3
Co-culture of E. coli and P. vulgatus
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Reference strains used for the tests were Escherichia coli ATCC 25,922 (Thermo Scientific) and Phocaeicola vulgatus ATCC 8482 (ATCC). Before assembling the co-culture system, bacteria were kept at -80ºC in Brain Heart Infusion Broth with glycerol (30%). At first, bacteria were inoculated on agar. Specifically, Trypton Soy Agar (TSA) (Sigma-Aldrich) was used for Escherichia coli, while Trypton Soy Agar supplemented with Defibrinated Sheep blood (5%) (Liofilchem) was used for Phocaeicola vulgatus. Both strains were cultivated for 24 h at 37ºC; Escherichia coli were cultured under aerobic conditions, while anaerobic conditions were used for Phocaeicola vulgatus. Bacterial suspensions were prepared using phosphate-buffered saline solution (Invitrogen).
4
Integrin-Mediated Cell Adhesion Assay
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Gelatin (denatured collagen I, from bovine skin), vitronectin (from human plasma), bovine serum albumin (BSA, fraction V) and Efavirenz were obtained from Sigma-Aldrich (Milan, Italy). Human recombinant IL-1β, TNF-α, and IFN-γ, and fibronectin from human plasma were purchased from Roche Molecular Biochemicals (Indianapolis, IN, USA). MAbs directed against the α5β1, αvβ3, αvβ5, or α6β4 integrins were obtained from Chemicon-Merck-Millipore (Darmstadt, Germany). The anti-CD31 (PECAM-1) mAb was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The Tat peptides (11–25), (46–60), and (66–80) were from UFP Service, University of Ferrara, Italy. Phosphate-buffered saline (PBS) solution, cell growth medium (RPMI 1640) and media supplements were obtained from Invitrogen-Life Technologies (Milan, Italy). Fetal bovine serum (FBS) was from HyClone (Logan, UT, USA).
5
Peptide-based Nanoparticles for Cell Studies
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Polymyxin B sulfate salt, calcein AM, ethidium homodimer-1 (EthD-1), dimethyl sulfoxide (DMSO), sodium hydroxide (NaOH), and hydrochloric acid (HCl, 37 wt%) were acquired from Sigma-Aldrich and used directly. Fluorescein isothiocyanate was provided by Thermo Fisher and used as received. Two amphiphilic peptides (Nap-Gly-Phe-Phe-Phe-Gly-Val-Asp-OH and Nap-Gly-Phe-Phe-Phe-Gly-Val-CONH2) were designed according to our previously studies (Xu et al., 2011 (link), 2012 (link)) and synthesized by GL Biochem Ltd. (Shanghai, China). Phosphate buffered saline (PBS) solution, Dulbecco’s Modified Eagle’s Medium (DMEM), luria broth (LB) medium, fetal bovine serum (FBS), penicillin-streptomycin, trypsin, and were provided by GIBCO Invitrogen Co. All other reagents and solvents were of analytical grade and used directly.
6
Fabrication of Culture Substrates
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The materials used for culture substrate fabrication were obtained from the following commercial sources: single-needle (glass; diameter, 600 μm; length, 3.2 cm) from Hirschmann Laborgeräte (Eberstadt, Germany); multi-needle (stainless steel; 9 needles; diameter, 500 μm; pitch, 500 μm; length, 1 cm) from Musashi Engineering, Inc., (Tokyo, Japan); synthetic oligopeptide CGGGKEKEKEKGRGDSP from Sigma-Aldrich (St. Louis, MO, USA); and poly(methyl methacrylate) plates from Comnet (Koube, Japan). The cells and regents used for cell culture were as follows: human umbilical vein endothelial cells (HUVECs, CC-2517A) from Cambrex Bio Science (Walkersville, MD, USA); immortalized HUVECs constitutively expressing green fluorescent protein (GFP-HUVECs), a generous gift from Dr. J. Folkman (Boston Children’s Hospital, Boston, MA, USA); endothelial basal medium (EBM-2, CC-3156) and SingleQuots growth supplement (CC-4176) from Lonza (Basel, Switzerland); type I collagen (Cellmatrix Type I-A) from Nitta Gelatin (Yao, Japan); Ham’s F12 medium and phosphate-buffered saline (PBS) solution from Invitrogen (Waltham, MA, USA); and PMA from Sigma-Aldrich. All other chemicals were purchased from Wako Pure Chemicals Industries (Odawara, Japan), unless otherwise indicated.
7
Recombinant HIV-1 Tat Protein Analysis
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Recombinant Tat protein from HIV-1 (subtype B) was expressed, purified, handled and tested for biological activity as described earlier (8 (link)). The human cervical cancer cell line SiHa harbouring HR-HPV16 DNA was cultured according to the supplier's protocol (American Type Culture Collection, Manassas, VA, USA). Growth medium (Dulbecco's modified Eagle's medium), supplements, phosphate-buffered saline (PBS) solution and the monoclonal antibody directed against the E7 protein of HPV16 were obtained from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Monoclonal antibodies raised against the E6 protein of HPV16, human p53 (clone DO-1) and human p16ink4a were acquired from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Anti-β-actin monoclonal antibodies, human plasma fibronectin (FN), bovine serum albumin (BSA, fraction V), doxorubicin hydrochloride and the chemicals employed for protein extraction were purchased from Sigma-Aldrich (St. Louis, MO, USA). The primers and probes employed for RNA analyses were purchased from Applied Biosystems (Thermo Fisher Scientific, Inc.).
8
Immunohistochemical Analysis of Oxidative Stress Markers
9
Cytotoxicity Evaluation of Hb-AuNCs
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Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Waltham, MA, USA) with 10% (v/v) fetal bovine serum (FBS) (Gibco, Waltham, MA, USA) was applied to culture Vero cells. The cytotoxicity of Hb-AuNCs was measured before and after incubation with Vero cells by a resazurin dye reduction assay. Vero cells were cultured in a 96-well plate (104 cells/well) for 24 h. After 24 h, the media in the 96-well plate were removed, and then a phosphate-buffered saline (PBS) solution (Thermo Fisher Scientific, Waltham, MA, USA) was utilized to wash each well cultured with Vero cells. Sequentially, Vero cells in the 96-well plates were incubated with Hb-AuNCs dispersed in DMEM. Concentrations of Hb-AuNCs of 2.7, 5.5, 11.0, 21.9, 43.8, 87.5, 175, 350, and 700 μg/mL were used to test the cytotoxicity. After 24 h of incubation, resazurin (0.02 mg/mL) (Thermo Fisher Scientific, Waltham, MA, USA) was added to each well and further incubated for an additional 4 h. The absorbances at 570 and 600 nm were measured by a plate reader (Thermo Varioskan Flash, Waltham, MA, USA). For the cell viability assay, each concentration of Hb-AuNCs was repeated independently at least eight times.
10
Optimizing Microscopy Imaging Techniques
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Example 13
Image acquisition protocol was varied extensively to fit the needs of each experiment. Snapshots, time-lapse, mosaics and z-stacks were all acquired on an Axio Observer Z1 inverted motorized microscope (Carl Zeiss, Germany) equipped with epifluorescence (Collibri™ 2), incubation chamber and Orcaflash V4.0 CMOS camera. Most images were acquired with a 10×, 0.45 NA, phase1 objective.
After cell culture for varying periods of time, HAHL-device was fixed for later staining and imaging. First, two washes were carried out by immersing disassembled device 5 min in phosphate-buffered saline (PBS) solution (ThermoFischer scientific). Then, the device was submerged in 4% (w/v), pH 7.4 paraformaldehyde (Sigma Aldrich, MI, US) solution for 15 minutes. Final two PBS washes were carried out before the device was sealed in a petri dish and kept at 4° C.
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