In vitro anti-OP effect by RBC-NPs was investigated based on the AChE activity on RBC ghosts following co-incubation with RBC-NPs and DDVP. Briefly, 100 μL of PBS (1×, pH = 7.2) solution containing 2 μL of RBC ghosts and different concentrations of RBC-NPs was incubated with different concentrations of DDVP for 30 min. Each sample then was centrifuged at 2,000 rpm in a Beckman Coulter Microfuge 22R Centrifuge for 10 min to selectively spin down the RBC ghosts, leaving RBC-NPs and DDVP in the supernatant. After discarding the supernatant, the pellet of RBC ghosts was suspended in 10 μL of PBS and their AChE activity was measured by an Amplex Red ACh/AChE assay kit (Invitrogen). To compare the AChE protection effect by different nanoformulations, 100 μL of PBS (1×, pH = 7.2) solution containing 4 mg/mL of RBC-NPs, PLGA-PEG NPs or PEG-liposomes was incubated with 10 μL of solution containing 5 μg DDVP for 30 min. Each sample was spun down at 14,000 rpm in a Beckman Coulter Microfuge 22R Centrifuge for 10 min to remove the nanoformulations. The supernatant was added to 2 μL of RBC ghosts and incubated for 30 min. AchE activity on the isolated RBC ghosts was measured as described above.
Microfuge 22r centrifuge
Manufactured by Beckman Coulter
Sourced in United States, Germany
The Microfuge 22R Centrifuge is a benchtop centrifuge designed for general laboratory applications. It features a maximum speed of 22,000 rpm and can accommodate a variety of rotor configurations to support different sample volumes and tube sizes.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay, by Thermo Fisher Scientific (4 mentions)
175 cm2 flask, by Corning (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Qiazol, by Qiagen (3 mentions)
Edta free 100x mammalian protease arrest, by G Biosciences (2 mentions)
M per, by Thermo Fisher Scientific (2 mentions)
K3 edta tubes, by Greiner (2 mentions)
F301.5 rotor, by Beckman Coulter (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Mini beadbeater, by Biospec (1 mentions)
Filtropur s, by Sarstedt (1 mentions)
Turbovap classic lv, by Biotage (1 mentions)
Amplex red ach ache assay kit, by Thermo Fisher Scientific (1 mentions)
Rnalater, by Merck Group (1 mentions)
Polytron, by Kinematica (1 mentions)
Adenosine triphosphate (atp), by Cayman Chemical (1 mentions)
Powergen 500, by Thermo Fisher Scientific (1 mentions)
Rocking platform shaker, by Avantor (1 mentions)
Chemdraw 12, by PerkinElmer (1 mentions)
41 protocols using microfuge 22r centrifuge
1
In vitro Anti-OP Activity of RBC-NPs
2
Caffeine Extraction from Water and Tissue
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Samples of water were defrosted at room temperature. Then, 5 mL was passed through a 0.45 μm syringe filter Filtropur S (Sarstedt, Nümbrecht, Germany) into 10 mL autosampler glass vials, and 5 ng of internal standard was added. For the tissue samples, fish were defrosted at room temperature, and then 0.1 g of muscle tissue from dorsal part of the body and whole brain were sampled into 2 mL polypropylene tubes from each fish. To extract caffeine from tissue samples, 1.5 mL of acetonitrile was added to the tubes together with 10 zirconium beads and samples were spiked with 50 ng of internal standard. Homogenization at 42000 oscillations per minute (Mini Beadbeater, BioSpec Bartlesville, USA) and centrifugation at 17500g for 10 min (Beckman Coulter Microfuge 22R Centrifuge) followed. The supernatant was pipetted into the 12-mL glass vial and whole extraction process was repeated with new 1.5 mL acetonitrile. The supernatants from both extractions were combined, evaporated to near dryness (TurboVap Classic LV, Biotage AB, Sweden), the eluent was reconstituted with 150 μL of methanol, and transferred into the 2 mL autosampler vials equipped with 200 μL inserts. The final extracts were frozen for a minimum of 24 h to ensure protein precipitation and centrifuged again directly before analysis.
3
Isolation and Assay of Cell Organelles
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The isolation
and assays were
performed similarly to that previously described.62 (link),63 (link) In brief, cells were grown to confluency with complete medium in
a 175 cm2 flask (Corning) before growth media was removed
and replaced with serum-free DMEM for 2 h. To neutralize endosomes
prior to lysis, FCCP was added to the cellular media to reach a final
concentration of 1 μM. Cells were incubated with FCCP for 15
min before cells were scrapped from the plate and pelleted at 1000g for 5 min. The media was discarded and cells were resuspended
in HEK assay buffer (20 mM HEPES, 5 mM glucose, 50 mM sucrose, 50
mM KCl, 90 mM potassium gluconate, 1 mM EGTA, Pierce protease inhibitor
mini tablet, pH = 7.4) and were then lysed by passage through a 22
gauge needle 10–15 times. Lysates were then centrifuged at
10,000g for 30 s with a Beckman Coulter Microfuge
22R centrifuge. The supernatant was removed and centrifuged at 14,500g for 20 min. The remaining supernatant was discarded, and
the pellet was resuspended in HEK assay buffer.
and assays were
performed similarly to that previously described.62 (link),63 (link) In brief, cells were grown to confluency with complete medium in
a 175 cm2 flask (Corning) before growth media was removed
and replaced with serum-free DMEM for 2 h. To neutralize endosomes
prior to lysis, FCCP was added to the cellular media to reach a final
concentration of 1 μM. Cells were incubated with FCCP for 15
min before cells were scrapped from the plate and pelleted at 1000g for 5 min. The media was discarded and cells were resuspended
in HEK assay buffer (20 mM HEPES, 5 mM glucose, 50 mM sucrose, 50
mM KCl, 90 mM potassium gluconate, 1 mM EGTA, Pierce protease inhibitor
mini tablet, pH = 7.4) and were then lysed by passage through a 22
gauge needle 10–15 times. Lysates were then centrifuged at
10,000g for 30 s with a Beckman Coulter Microfuge
22R centrifuge. The supernatant was removed and centrifuged at 14,500g for 20 min. The remaining supernatant was discarded, and
the pellet was resuspended in HEK assay buffer.
4
Isolation of Cardiac Mitochondria for RNA Analysis
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Freshly isolated mitochondria were prepared from hearts after perfusion with RNAlater (Sigma‐Aldrich, St. Louis, MO), by differential centrifugation.15 Briefly, at the end of perfusion, the left ventricle was dissected out and placed in Buffer A (in mmol/L: 180 KCl, 2 EGTA, 5 MOPS, 0.2% BSA; pH 7.25). The tissue was then digested with trypsin (0.0001/0.1 g tissue) in 0.7 mL of ice‐cold Buffer B (in mmol/L: 225 Mannitol, 75 sucrose, 5 MOPS, 0.5 EGTA, 2 taurine; pH 7.25) and finally homogenized with Buffer B with a protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN) using a Polytron (Kinematica, Bohemia, NY). To further separate the heart mitochondria from other cellular components and tissue debris, a series of differential centrifugations were performed in a Microfuge 22R centrifuge (Beckman Coulter, Fullerton, CA) at 4°C. The crude pellet was then lysed with QIAzol (Qiagen, Valencia, CA).12
5
Enzymatic Characterization of TPK1
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The functionality of TPK1 was determined by an ex vitro enzymatic assay [56 (link)]. TPK1 catalyzes the transfer of two phosphate groups onto thiamine in the presence of adenosine triphosphate (ATP) and Mg2+. Cell lysates were prepared by washing treated cells with ice-cold PBS followed by lysis using mammalian protein extraction reagent (M-PER, Thermo Scientific, Rockford, IL) containing 10 μL/mL of EDTA free-100X Mammalian Protease Arrest (G-Biosciences, St. Louis, MO). Lysates were collected and centrifuged at 17,000 × g using a Microfuge 22R Centrifuge (Beckman Coulter, Brea, CA) for 10 min at 4° C to pellet cellular debris. Protein concentration of the resulting supernatant were quantitated by BCA protein assay (Thermo Scientific, Rockford, IL). Lysates (0.5 mg total protein) were then combined with ATP (5 mM, disodium salt, Cayman Chemical, Ann Arbor, MI) and MgSO4 (10 mM) in 0.02 M Tris-HCl reaction buffer (pH8.6). Thiamine (10 μM) was added to initiate the TPK1 reaction, which was allowed to proceed at 37° C for 30 min. To stop the reaction, proteins were precipitated by the addition of 10% trichloroacetic acid and the mixture was immediately placed on ice. The extent of TPP produced was determined via HPLC as described above.
6
Hippocampal Lipid Extraction Protocol
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The extraction protocol was adapted from a previously described method [29 (link)]. Hippocampal weights (~10 mg) were measured and the tissue mixed with methanol/MTBE (400 μL, 1:3, v/v) before homogenization (PowerGen 500, Fisher Scientific, San Diego, CA, USA). Samples were then probe sonicated for 20 s, 2 Watts (Misonix, Farmingdale, NY, USA), and shaken (Rocking Platform Shaker, VWR International, USA) for 20 min at 4 °C. Afterward, a methanol/water mixture (400 μL, 1:3, v/v) was added, and samples were vortexed for 10 s and centrifuged for 7 min (4 °C, 14,000 rpm), using a Microfuge 22R Centrifuge (Beckman Coulter, Brea, CA). Supernatant (150 μL) was collected, dried under argon, and stored at −80 °C until analysis.
7
Isolation of Heart Mitochondria
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Freshly isolated mitochondria were prepared from hearts after perfusion with RNAlater, by differential centrifugation [11] (link). Briefly, at the end of perfusion, the left ventricle was dissected out and placed in Buffer A (in mM: 180 KCl, 2 EGTA, 5 MOPS; 0.2% BSA; pH: 7.25). The tissue was then digested with trypsin (0.0001 g/0.1 g tissue) in 0.7 ml of ice-cold Buffer B (in mM: 225 Mannitol, 75 sucrose, 5 MOPS, 0.5 EGTA, 2 Taurine; pH: 7.25) and finally homogenized with Buffer B with a protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN) using a Polytron. To further separate the heart mitochondria from other cellular components and tissue debris, a series of differential centrifugations were performed in a Microfuge 22R centrifuge (Beckman Coulter, Fullerton, CA) at 4°C. The crude pellet was then lysed with QIAzol (Qiagen, Valencia, CA) [11] (link).
8
Metabolite Profiling by HPLC-MALDI-TOF MS
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EtOH (50% in MQ)-extract was separated with the HPLC method and as a parallel assay, the fraction related to the corresponding peaks on a chromatogram was collected in a test tube for further MALDI-TOF MS detection. EtOH (50% in MQ)-extracts were centrifuged with Beckman Coulter Microfuge® 22R Centrifuge under the room temperature at the rate of 14.000 rpm for 7 min. Then, the pellet was eliminated, and the supernatant was utilized to perform HPLC analysis.
All structures were obtained using ChemDraw 12.0 (PerkinElmer) software and the MALDI-TOF MS and MS/MS spectra were processed with Daltonics flexAnalysis 3.4 (Bruker) software. Chromatograms were obtained with Milichrom A-02 (Econova) software utilization and processed with Multichrom for Windows 9x&NT version 1.5x-E” (Ampersend, Moscow, Russia) software.
All structures were obtained using ChemDraw 12.0 (PerkinElmer) software and the MALDI-TOF MS and MS/MS spectra were processed with Daltonics flexAnalysis 3.4 (Bruker) software. Chromatograms were obtained with Milichrom A-02 (Econova) software utilization and processed with Multichrom for Windows 9x&NT version 1.5x-E” (Ampersend, Moscow, Russia) software.
9
Quantitative Cytoplasmic Histone-DNA Fragmentation
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For quantitative determination of cytoplasmic histone-associated DNA fragments, the Cell Death Detection ELISAPLUS (Roche LifeScience, Indianapolis, IN) was utilized following the manufacturer’s instructions. Briefly, cells were lysed in buffer supplied by the kit for 30 min at room temperature with shaking. Lysates were collected and centrifuged at 200xg for 10 min at 4°C in a Microfuge 22R Centrifuge (Beckman Coulter). Supernatant was added to the supplied microplate in duplicate along with Immunoreagent for 2h at room temperature with shaking at 300rpm. At the end of the incubation period, the solution was removed and plates were washed 3 times each with incubation buffer. Additionally, ABTS solution was added to each well and left for 10 min at room temperature with shaking at 250rpm. Finally, ABTS stop solution was added, and the absorbance at 405nm was measured using a SpectraMax M2 spectrophotometer (Molecular Devices; Sunnyvale, CA).
10
Isolation of Bacteria from Sea Urchin Gonads
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The bacteria from sea urchin gonads were collected according to Chen et al. (2019) (link). The sea urchin gonads (10 g) were homogenized with normal sterile saline (95 mL), and centrifuged at 2600× g at 4 °C for 5 min (Microfuge 22R Centrifuge, Beckman Coulter Inc., MA, USA). And then, the supernatant was separated and centrifuged again at 8800× g for 5 min. The bacteria were collected from the precipitate.
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