For the proteasomal activity assay, we lysed cells with a buffer containing 40 mM TRIS pH 7.2 (Merck), 50 mM NaCl (Merck), 5 mM MgCl2(hexahydrate) (Merck) and 10% vol/vol glycerol (Sigma-Aldrich) freshly supplemented with 2 mM β-mercaptoethanol (Sigma-Aldrich) and 2 mM ATP (Sigma-Aldrich). To measure chemotrypsin-like, trypsin-like and caspase-like activity, we used the Proteasome Activity Fluorometric Assay (UBP Bio) according to the manufacturer’s instructions. For the normalization of proteasomal activity to total protein content, we used the Bio-Rad Protein Assay Kit II to determine protein concentration in our samples.
Tris ph 7
Manufactured by Merck Group
Sourced in Japan
Tris pH 7.5 is a buffer solution that maintains a pH of 7.5. It is commonly used in various laboratory applications to maintain a stable pH environment.
Automatically generated - may contain errors
Lab products found in correlation
Sodium chloride (nacl), by Merck Group (4 mentions)
Adenosine triphosphate (atp), by Merck Group (2 mentions)
Glycerol, by Merck Group (2 mentions)
Mgcl2, by Merck Group (2 mentions)
Protein assay kit 2, by Bio-Rad (2 mentions)
Ficoll pm 400, by Merck Group (2 mentions)
0.5m edta, by Thermo Fisher Scientific (2 mentions)
Sarkosyl, by Teknova (2 mentions)
Hw 65s, by Merck Group (2 mentions)
Dithiothreitol (dtt), by Teknova (2 mentions)
Autocad, by Autodesk (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (2 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Normal melting point agarose, by Merck Group (1 mentions)
Low melting point agarose, by Merck Group (1 mentions)
Dm4000 microscope, by Leica camera (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
11 protocols using tris ph 7
1
Proteasomal Activity Assay Protocol
2
Proteasomal Activity Quantification
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Proteasome activity To prepare lysates for the proteasomal activity assay, cells were lysed in lysis buffer (40 mM TRIS pH 7.2 (Merck), 50 nM NaCl (Merck), 5 mM MgCl2(hexahydrate) (Merck), 10 % v/v glycerol (Sigma), 2 mM ATP (Sigma), 2 mM 2-mercaptoethanol (Sigma). Activity was measured using the Proteasome Activity Fluorometric Assay II kit (UBPBio, J41110), according to manufacturer's instructions in a Tecan Plate reader. This assay allowed for measurements of chymotrypsin-like, trypsin-like, and caspase-like activity. The results were then normalized to either protein, using Bio-Rad Protein Assay Kit II (Bio-Rad), or DNA, using the Quant-iT PicoGreen dsDNA assay kit (Invitrogen, P7589), both according to manufacturer's instructions.
3
Comet Assay for Genotoxicity Evaluation
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The SCGE assay was performed as described by Tice & Vasquez with some modifications. The remaining 20 μL of cells was placed in 150 μL of low melting point agarose (0.5%; Sigma, St. Louis, MO) and spread onto two microscope slides that had been prelayered with 180 μL of normal melting point agarose (1%; Sigma, St. Louis, MO); the slides were then immersed in a chilled lysis solution (2.5 M·NaCl, 100 mM Na2EDTA, 10 mM Tris at pH 10, 10% DMSO, 1% Triton X-100 fresh; Sigma, St. Louis, MO). After lysis at 4 °C for 1 h, the slides were placed on a horizontal electrophoresis unit (Gibco BRL Life Technologies Inc.). The DNA was allowed to unwind for 20 min in electrophoresis running buffer solution (300 mM·NaOH, 1 mM·Na2EDTA at pH >13; Sigma, St. Louis, MO). Electrophoresis was conducted for 20 min at 25 V and 300 mA (0.73 V/cm). All the technical steps were carried out under very dim indirect lighting. After electrophoresis, the slides were gently removed and rinsed with neutralization buffer (0.4 M·Tris pH 7.5, Sigma, St. Louis, MO); they were then dehydrated with ethanol (70%) and air-dried. All of the slides were coded before analysis. Ethidium bromide (75 μL of 20 μg/mL solution) was added to each slide, and a cover glass was placed on the gel. Individual nuclei were visualized under 400x magnification on a Leica DM4000 microscope.
4
Microfluidic Device Design and Fabrication
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Microfluidic devices were designed using AutoCAD (AutoDESK, USA), tested using COMSOL Multiphysics as well as
empirically, and fabricated using soft lithographic techniques22 (link) (Supplementary Data 1 ). The devices were tested on a Drop-seq setup,
using bare beads (Tosoh, Japan, Cat # DTTiCZIBPBHhf-iF1YcK/" target="_blank">HW-65s) in Drop-Seq Lysis Buffer (DLB7 (link); 10 ml stock consists of 4 ml of nuclease-free H2O, 3 ml 20% Ficoll PM−400
(Sigma, Cat # F5415-50ML), 100 μl 20% Sarkosyl (Teknova, Cat # S3377), 400 μl 0.5M
EDTA (Life Technologies), 2 ml 1M Tris pH 7.5 (Sigma), and 500 μl 1M DTT (Teknova, Cat # D9750), where the DTT
is added fresh) and 1x PBS, to optimize flow and bead occupancy parameters in drops. Droplet generation was assessed under a
microscope in real time using a fast camera (Photron, Model # SA5), and later by sampling the emulsion using a
disposable hemocytometer (Life Technologies, Cat # 22-600-100) to check droplet integrity, size, and bead occupancy.
The device design is provided as aSupplementary File 1 and Supplementary Fig. 1a . The unit in the CAD provided is 1 unit =
1 μm; channel depth on device is 75 μm.
empirically, and fabricated using soft lithographic techniques22 (link) (
using bare beads (Tosoh, Japan, Cat # DTTiCZIBPBHhf-iF1YcK/" target="_blank">HW-65s) in Drop-Seq Lysis Buffer (DLB7 (link); 10 ml stock consists of 4 ml of nuclease-free H2O, 3 ml 20% Ficoll PM−400
(Sigma, Cat # F5415-50ML), 100 μl 20% Sarkosyl (Teknova, Cat # S3377), 400 μl 0.5M
EDTA (Life Technologies), 2 ml 1M Tris pH 7.5 (Sigma), and 500 μl 1M DTT (Teknova, Cat # D9750), where the DTT
is added fresh) and 1x PBS, to optimize flow and bead occupancy parameters in drops. Droplet generation was assessed under a
microscope in real time using a fast camera (Photron, Model # SA5), and later by sampling the emulsion using a
disposable hemocytometer (Life Technologies, Cat # 22-600-100) to check droplet integrity, size, and bead occupancy.
The device design is provided as a
1 μm; channel depth on device is 75 μm.
5
Synchronize Cells for FISH Experiments
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FACS was used to derive live cells with uniform mEGFP expression. Cells were sorted in PBS supplemented with 2% (vol/vol) FBS and 2 mM EDTA (AppliChem) in a FACSAria III (BD) flow cytometer. To assess cell synchronization for FISH experiments, DNA content was measured in cells detached from the plate surface using 0.25% trypsin (Thermo Fisher Scientific), washed twice with PBS, and fixed with 73% ice-cold methanol (Sigma-Aldrich) at −20°C overnight. The next day, cells were washed with PBS and stained for 30 min at 37°C in a propidium iodide buffer containing 50 µg/ml propidium iodide (Sigma-Aldrich), 10 mM Tris, pH 7.5 (Sigma-Aldrich), 5 mM MgCl2 (Sigma-Aldrich), and 200 µg RNase A (Thermo Fisher Scientific). Cell cycle profiles were analyzed with a FACSCanto flow cytometer (BD).
6
Microfluidic Device Design and Fabrication
7
Immunoprecipitation and Western Blotting
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Cells were lysed with IP buffer (10 mM Tris pH7.4 (Sigma-Aldrich), 150 mM NaCl (Sigma-Aldrich), 1% NP-40 (USB Corporation), Proteinase inhibitor cocktail (Roche), 1 mM NaF, 1 mM Na3VO4 (Sigma-Aldrich) and PMSF (Sigma-Aldrich) in deionized H2O) for 30 minutes at 4°C. The supernatant was collected by centrifugation at 16,000g for 5 minutes at 4°C. In some experiments, cell extracts were cleared by a 200,000g ultracentrifugation. After preclearing with protein A/G beads for 1 hour, cell lysates were incubated with HA-EZ Agarose Beads (Sigma-Aldrich) for 3 hours or anti-Myc primary antibody for 3 hours followed by incubation with protein A/G beads for 1 hour at 4°C with gentle agitation. The beads were then extensively washed with IP buffer and boiled for 10 minutes at 95oC in SDS loading buffer for WB analysis.
8
DOTA Conjugation of Anti-Mouse TREM1 Antibody
9
AADC Enzyme Activity Assay in Neuronal Cultures
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AADC enzyme assay was performed using the refined method developed in Allen24 from Hyland and Clayton.25 (link) Neuronal cultures at Day 65 in phenol red-free medium were harvested and lysed by snap freezing twice in liquid nitrogen in 100 µl of 10 mM Tris pH 7.4 (Sigma-Aldrich), 1 mM EDTA, 320 mM sucrose (Sigma-Aldrich) and protease inhibitor cocktail (Roche). Cell lysate (50 μl) was incubated with 70 μM pyridoxal 5′-phosphate (PLP, Sigma-Aldrich) in assay buffer composed by 500 mM sodium phosphate pH 7.0, 0.167 mM EDTA, and 39 mM dithiothreitol (Sigma-Aldrich) for 120 min at 37°C, and subsequently 2 mM final concentration of l -DOPA (Sigma-Aldrich) was added and incubated for 20 min at 37°C. The reaction was stopped with 250 μl of 0.8 M perchloric acid (final concentration 0.4 M) for 10 min at room temperature and centrifuged at 12 000g for 5 min at 4°C. A substrate blank with no l -DOPA and a sample blank without cell lysate were performed for each sample. Dopamine in the supernatant was then quantified by high performance liquid chromatography (HPLC).
10
Quantifying Asexual Parasite Growth
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Asexual parasite blood stage growth (measuring the ability of parasites to invade, replicate, exit and reinvade erythrocytes) was undertaken using the SYBR Green asexual growth assay, undertaken largely as published55 (link). In brief 96-well black clear bottom plates (Corning) were pre-printed with test compound and normalized with DMSO (Merck) to 0.5% of a total assay volume of 100 μl. Highly synchronized ring stage parasites and blood was added to each well so that the final parasitaemia was 2% and the hematocrit was 1%. The compound was incubated with parasites for 72 h before being frozen at −20 °C (to aid with cell lysis). The plate was thawed on ice and a lysis buffer (20 mM Tris pH7.5 (Merck), 5 mM EDTA (Merck), 0.008% w/vol saponin (Merck), 0.08% vol/vol triton-x100 (Merck) and SYBR-Green I (Thermo Fisher Scientific) at a final concentration of 0.02% vol/vol). The 96-well plate was incubated for 1 h at RTP before each well was assayed for fluorescence using GFP filters (Excitation 485 nm/Emission 535 nm) on a microplate reader (TECAN).
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