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Phosphate buffer

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Phosphate buffer is a solution used to maintain a stable pH in various laboratory applications. It is composed of a mixture of sodium phosphate and potassium phosphate salts. The buffer helps to maintain a consistent pH environment, which is essential for many chemical and biological processes.

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32 protocols using phosphate buffer

1

Fixation and TEM Imaging of AML and MDM Cells

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AML and MDM cells were fixed with 4% formaldehyde and 1% glutaraldehyde in phosphate buffer (Invitrogen) overnight at 4°C. The samples were processed and imaged using a JEOL 1400 TEM. Expanded protocol is given in the detailed supplemental methods section.
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2

Inhibition of Amyloid-Beta Aggregation

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1–40 and Aβ1–42 peptides were purchased from American Peptide (Sunnyvale, CA). Peptides were solubilized in HFIP (Sigma), and dried overnight [27] (link). Compound stock was dissolved in phosphate buffer (pH 7.4, Invitrogen) at 200 μM. Aβ1–40 and Aβ1–42 (100 μg/ml in PBS) were mixed with different concentration of compound at 1∶1 volume and incubated at 37°C for 24 hours. The effect of compound on Aβ aggregation was analyzed by western blot analysis using 6E10 antibody. Two β-adrenergic receptor blockers, Carvedilol, which strongly inhibits Aβ aggregation, and Acebutolol, which has no effect on Aβ aggregation, were used as positive and negative controls in the aggregation assay.
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3

Micronucleus Assay Protocol for Bone Marrow

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The MN assay followed the protocol described elsewhere (37 (link), 38 (link)). Bone marrow cells were harvested using foetal calf serum (2 mL) 24 h after receipt of the last dose. Cell pellets obtained by centrifugation at 300 g for 5 min were then dissolved again in about 500 μL of foetal calf serum.
Two smears were prepared for each treatment and air-dried prior to fixing in 90 % methanol at -20 °C for 20 min and staining with acridine orange (MP Biomedicals) for 2 min. After washing with phosphate buffer (Invitrogen, Carlsbad, CA, USA) twice for 3 min each, two slides per dose group were coded and scored blindly for MN in about 1000 reticulocytes (RETs) or polychromatic erythrocytes (PCEs) per slide at 1000x magnification under UV light using an Olympus BX50 fluorescent microscope (Southend-On-Sea, UK). We also determined the percentage of RETs or PCEs/normochromatic erythrocytes (NCEs) per 1000 cells, as any reduction in the number of PCEs or RETs is a sign of bone marrow toxicity.
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4

Ultrastructural Analysis of AML and MDM

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AML and MDM cells were fixed with 4% formaldehyde and 1% glutaraldehyde in phosphate buffer (Invitrogen) overnight at 4°C. The samples were processed and imaged using a JEOL 1400 TEM. Expanded protocol is in the detailed supplemental methods section.
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5

Anterograde Tracing of Prefrontal Connections

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The training and validation datasets came from two different male rhesus macaques. The former received an injection of the anterograde/bidirectional tracer Lucifer Yellow in the anterior frontal cortex (frontal pole). The latter received an injection of the anterograde/bidirectional tracer Fluorescein in the ventrolateral prefrontal cortex (vlPFC). Surgery and tissue preparation were performed at the University of Rochester Medical Center. Details of these procedures were described previously (Haber, 1988 (link); Lehman et al., 2011 (link); Safadi et al., 2018 (link)). Briefly, each monkey received an injection of a bidirectional tracer conjugated with dextran amine (40–50 nl, 10% in 0.1 M phosphate buffer, pH 7.4; Invitrogen). Twelve days after the injection, animals were perfused and their brains were postfixed overnight and cryoprotected in increasing gradients of sucrose (10, 20, and 30%). All experiments were performed in accordance with the Institute of Laboratory Animal Resources Guide for the Care and Use of Laboratory Animals and approved by the University of Rochester Committee on Animal Resources.
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6

Evaluating P-gp Inhibition and Cell Viability

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PTX and FKA were purchased from Sigma (St. Louis, MO, USA). Sodium aescinate (Aes) (≥98%) was purchased from Meilunbio® (Dalian, China). Monoclonal rabbit anti-human P-gp (cat. no. 13978) was purchased from Cell Signaling Technology (MA, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies (Kumamoto, Japan). Annexin V‑fluorescein isothiocyanate/propidium iodide Apoptosis Detection Kit was purchased from BD Biosciences (Franklin Lakes, NJ, USA). The cell cycle detection kit was purchased from Sigma (St. Louis, MO, USA). Dialysis membranes (MWCO 1000) were obtained from the Shanghai Medical Chemical Reagent Co., Ltd. (Shanghai, China). Phosphate buffer and 0.25% trypsin were purchased from Gibco, Invitrogen Corp (Ontario, USA).
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7

Anterograde Tracing of Prefrontal Connections

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The training and validation datasets came from two different male rhesus macaques. The former received an injection of the anterograde/bidirectional tracer Lucifer Yellow in the anterior frontal cortex (frontal pole). The latter received an injection of the anterograde/bidirectional tracer Fluorescein in the ventrolateral prefrontal cortex (vlPFC). Surgery and tissue preparation were performed at the University of Rochester Medical Center. Details of these procedures were described previously (Haber, 1988 (link); Lehman et al., 2011 (link); Safadi et al., 2018 (link)). Briefly, each monkey received an injection of a bidirectional tracer conjugated with dextran amine (40–50 nl, 10% in 0.1 M phosphate buffer, pH 7.4; Invitrogen). Twelve days after the injection, animals were perfused and their brains were postfixed overnight and cryoprotected in increasing gradients of sucrose (10, 20, and 30%). All experiments were performed in accordance with the Institute of Laboratory Animal Resources Guide for the Care and Use of Laboratory Animals and approved by the University of Rochester Committee on Animal Resources.
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8

Isolation and Culture of Cancer-Associated Fibroblasts

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The tissue material was first rinsed with 50% ethanol (Merck, Darmstadt, Germany), followed by phosphate buffer (Invitrogen, Waltham, MA, USA). The necrotic and adipose tissues were then removed from the tissue using a sterile scalpel, and the tumour tissue was cut into 3 mm pieces. Next, samples in the trypsin media were kept at 37 °C to activate the trypsin. Subsequently, the tumour pieces were transferred to a sterile tube with phosphate buffer and centrifuged at 4 °C, 2700 rpm for 7 min. After removing the supernatant, the tumour pieces were placed into the culture medium (MEM) with added 10% FBS (Biochrom, Holliston, MA, USA), 1% antibiotic-antimycotic solution, 10 μg/mL gentamicin sulfate, and 10 μg/mL ciprofloxacin (Santa Cruz Biotechnology, Dallas, TX, USA) to prevent contamination. After 7 days the culture medium was replaced with MEM medium with 10% FBS, supplemented with antibiotics (penicillin 100 U/mL and streptomycin 0.1 mg/mL) (Biochrom, Holliston, MA, USA) and 0.4 μg/mL hydrocortisone (Merck, Darmstadt, Germany). Since we were primarily interested in the effect of TME-derived fibroblasts (CAFs), selection using population overgrowth was performed. The passages of CAFs ranged from 1 to 5.
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9

Drug Stability and Metabolism Assays

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Drug stability in plasma was evaluated by incubating 5 μM PY10, PY109, PY108, or ITE in mouse plasma for 0, 5, 15, 30, 45, and 60 min at 37°C with constant shaking at 225 rpm. Reactions were terminated by the addition of twofold (v/v) acetonitrile, followed by centrifugation at 14,000 rpm for 15 min to remove particulates, and the supernatant was analyzed by LC-MS/MS.
Drug metabolic stability was examined using a liver microsomal assay (45 (link)). Briefly, 5 μM PY10, PY109, PY108, or ITE was added to pooled mouse liver microsomes (0.5 mg/ml) in 100 mM phosphate buffer containing MgCl2 (Thermo Fisher Scientific). The assay was activated by the addition of an NAPDH (reduced nicotinamide adenine dinucleotide phosphate)–regenerating system containing NADP+, glucose-6-phosphate, and glucose-6-phosphate dehydrogenase (Sigma-Aldrich). Samples were incubated for 0, 5, 15, 30, and 45 min at 37°C, with constant shaking (225 rpm). Reactions were terminated by adding twofold (v/v) acetonitrile, samples were centrifuged at 14,000 rpm for 15 min, and the supernatant was analyzed using LC-MS/MS. t1/2 values were calculated according to the equation t1/2=0.693/k where k is the elimination rate constant, obtained by fitting C = initial × exp.k × t.
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10

Quantifying Extracellular Hydrogen Peroxide

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Cells were exposed to DBD plasma and after 24h incubation at 37 °C, 50 μM Amplex® red reagent and 0.1 units/ml horseradish peroxidase in phosphate buffer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) were added to each well to a final volume of 100 μl, and the cells were incubated for 30 min in the dark. Fluorescence was assessed at excitation/emission values of 485 nm/580 nm in a microplate reader.
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