Institutional Review Board at the University of Iowa reviewed and approved human studies involving healthy donors (200911748) and exempted the studies involving blood from critically ill patients. Patients were screened for presence or absence of sepsis in the intensive care units at the University of Iowa (30 ). Venous blood was drawn into tubes coated with heparin and neutrophils were isolated within 3 hours of collection(31 (link)). Neutrophil subcellular fractions were obtained by discontinuous 2-layer Percoll density gradient centrifugation with a slight modification (32 (link)). Cells were incubated with 1 mM Diisopropylfluorophosphate (Sigma-Aldrich #D0879), an irreversible serine protease inhibitor, for 20 minutes on ice prior to cell disruption by nitrogen cavitation. Amounts of each subcellular component recovered are expressed as Cell Equivalent (CE) of starting neutrophil counts. Whole cell lysates were obtained by suspending cells in a buffer consisting of 25mM Tris pH 7.4, 150mM Sodium Chloride, 5% Glycerol, 1% NP-40 with Complete Mini (Roche) protease inhibitor cocktail and PhosStop (Roche) phosphatase inhibitor cocktail. Cells were disrupted with a probe sonicator, centrifuged at 16000×g for 10 minutes, and supernatants were stored −80°C until use.
Diisopropylfluorophosphate
Manufactured by Merck Group
Sourced in United States
Diisopropylfluorophosphate is a chemical compound used in various laboratory applications. It is a colorless, volatile liquid with a pungent odor. The core function of Diisopropylfluorophosphate is to act as a highly potent inhibitor of acetylcholinesterase, an enzyme involved in the breakdown of the neurotransmitter acetylcholine.
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Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Phosstop, by Roche (1 mentions)
Complete mini, by Roche (1 mentions)
Ponatinib, by Bio-Techne (1 mentions)
Microcystin lr, by Enzo Life Sciences (1 mentions)
Flag lrrk2 g2019s, by Thermo Fisher Scientific (1 mentions)
D0879, by Merck Group (1 mentions)
Atropine sulfate, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 cell medium, by Thermo Fisher Scientific (1 mentions)
Apoptotic dna ladder kit, by Roche (1 mentions)
2 4 dinitrophenylhydrazine, by Merck Group (1 mentions)
Enzchek caspase 3 assay kit, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Reduced glutathione, by Merck Group (1 mentions)
Anti puromycin, by Merck Group (1 mentions)
β actin clone ac 74, by Merck Group (1 mentions)
Stat2, by BD (1 mentions)
Clone 736e11, by Cell Signaling Technology (1 mentions)
15 protocols using diisopropylfluorophosphate
1
Neutrophil Subcellular Fractionation Protocol
2
Synthesis and Characterization of LRRK2 Modulators
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GSK3357679A [25 (link)]was synthesized at GlaxoSmithKline. MLi-2 was synthesized by Natalia Shpiro (University of Dundee) as described previously [16 (link)]. GZD-824 (#21508) and Rebastinib (#21465) were purchased from Cambridge Bioscience and Ponatinib (#4274) from Tocris. Microcystin-LR was purchased from Enzo Life Sciences (#ALX-350-012-M001). Human recombinant full length wild-type Flag-LRRK2 (#A15198) and Flag-LRRK2[G2019S] (#A15201) were from ThermoFisher Scientific. Diisopropylfluorophosphate was from Sigma (#D0879).
3
Neutrophil Activation and Protein Analysis
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For western blotting, 2x106 neutrophils pretreated with indicated HDAC inhibitor, or protein-arginine deaminase (PAD4) inhibitor GSK484, and then stimulated with 1 μM PMA for 3 hours were lysed using RIPA buffer with protease and phosphatase inhibitors (T-2494, A.G. Scientific) and diisopropylfluorophosphate (D0879, Sigma). Immunoblotting was performed using standard molecular biology techniques. Band intensity was quantified using ImageJ software.
4
Optimized DFP Toxicity Protocols
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Diisopropylfluorophosphate (Sigma-Aldrich, purity >97%) was prepared fresh in cold phosphate-buffered saline (PBS) prior to administration. Atropine sulfate (ATS, Thermo-Fisher Scientific) and 2-pralidoxime (2-PAM) were prepared fresh in saline. Midazolam (MDZ) was purchased from the Lloyd Veterinary Medical Center Hospital Pharmacy. Gelatin for tissue embedding consisted of 15% type A porcine gelatin, 7.5% sucrose, and 0.1% sodium azide. Citric acid buffer contained 10 mM citric acid and 0.05% tween-20 at a pH of 6. Blocking buffer consisted of 10% donkey serum and 0.05% tritonX-100 in PBS. Antibodies used for immunohistochemistry (IHC) are summarized in Supplementary Table S1 . All antibodies were tested with a negative control simultaneously, and the optimal concentration was determined by serial dilution. Antibodies were diluted in PBS containing 2.5% donkey serum, 0.1% tritonX-100 and 0.25% sodium azide. Streptavidin conjugated antibodies were diluted in PBS.
5
Preparation and Use of Hippocampal Slices
6
Optimized Apoptosis and Viability Assays
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Reduced glutathione (GSH), digitonin, dimethyl sulfoxide (DMSO), 2,2,2-trichloroacetic acid (TCA), 2,4-dinitrophenylhydrazine (DNPH), 5,5’-dithiobionitrobenzoic acid (DTNB) and diisopropyl fluorophosphate (DFP) were purchased from Sigma Chemical Company (St. Louis, MO). DMEM, KSFM and growth factors, and RPMI 1640 cell medium, penicillin/streptomycin solution, and geneticin (G418) and KB plus DNA ladder, Celltracker blue (7-amino-4-chloromethylcoumarin or CMAC), 10kD spin columns, and EnzChek Caspase-3 assay kit were purchased from Invitrogen (Grand Island, NY). BCA kit and the anti-DYKDDDDK (anti-FLAG) antibody (PA1-984B) were purchased from Pierce (Rockford, IL). Celltiter 96 AQueous One MTS kit, described as the MTS viability assay in experiments, was purchased from Promega (Madison, WI) and contained CellTiter96 Aqueous One Solution composed of a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfonyl)-2H-tetrazolium, inner salt (MTS) and an electron coupling reagent (phenazine methosulfate). The Apoptotic DNA ladder kit was purchased from Roche (Indianapolis, IN). All chemicals used for the synthesis of prodrugs were purchased from Sigma-Aldrich (St. Louis, MO), TCI (Portland, OR), Acros Organics (Thermo Fisher Scientific, New Jersey) and Lancaster (Ward Hill, MA) and used without further purification.
7
Immunoblot Analysis of Signaling Pathways
8
Acetylcholinesterase Inhibition Assay
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We purchase lyophilized Electrophorus electricus AChE, demeton-S-methyl, and diisopropylfluorophosphate from Sigma Aldrich. We run a modified protocol from Cherny et al.19 (link), where standard Ellman assays recover kinetic parameters26 (link) and determine the ability of Smu. 1393c to protect AChE from the effects of OP simulants. We incubate equal amounts of Smu. 1393c and OP simulant (final concentration 500 nM) at 20 °C for 30 minutes. We add AChE to each sample and incubate at 20 °C for 60 minutes. As a detectable marker we add 500 μM of the Ellman reagent 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) to the samples. We vary the amount of acetylthiolcholine (0–20 mM) and bring each sample to a volume of 50 μL using buffer (200 mM NaCl, 20 mM TRIS at pH 7.4). Each assay is performed in triplicate. We plot the kinetic parameters, obtained using SpectraMax M3 plate reader, and fit them to the Michaelis-Menton kinetic equation.
9
Subtilisin 72 Substrate Assay
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Subtilisin 72 [21 (link)], peptide substrates ZAAL-pNA (N-benzyloxycarbonyl-alanyl-alanyl-leucine p-nitroanilide) [22 ], ZAAR (N-benzyloxycarbonyl-alanyl-alanyl-arginine) [23 ], and ZAAL (N-benzyloxycarbonyl-alanyl-alanyl-leucine) [24 ] were prepared in our laboratory. Affinity sorbent ([N-(ε-amino-caproyl)-p-aminobenzyl]succinyl-Sepharose 4B (CABS-Sepharose) was prepared according to previously described protocols [25 (link)]. Ultrafiltration cells and PM10 membranes were from Amicon (Millipore-Sigma, Birlington, MA), glass capillaries 60-mm in length and inner diameter of 0.5 mm for CPT crystallization from Confocal Science Inc. (Tokyo, Japan). Deionized water with resistance of 19 MOhm/cm was prepared on the MilliQ system (Merck Millipore, Middlesex Turnpike Billerica, M A), 2-methyl-2,4-pentadiol (MPD), L-cystine and L-cysteine, diisopropyl fluorophosphate, isopropyl-β-D-thiogalactoside (IPTG), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and 4-morpholinoethansulfonic acid (MES), and porcine carboxypeptidase B were obtained from Sigma-Aldrich (St. Louis, MO). The kinetic experiments were performed in the thermostated cell of the Shimadzu UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan) connected with IBM PC.
10
Chitosan-Cyanamide-DFP Synthesis Protocol
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Chitosan (deacetylation degree: 77% based on the FT-IR spectrum [25 ] and 50,000–190,000 Da based on viscosity), cyanamide (99%), and diisopropylfluorophosphate (DFP) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetic acid (98%) and sodium hydroxide (93%) were purchased from Duksan Chemical Co., Ltd (Ansan, Korea). Hydrochloric acid (35–37%) and ethyl alcohol (99%) were purchased from Daejung Chemical Co., Ltd (Siheung, Korea).
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