In this study, 5 mL fasting blood samples were collected from the subjects in the UA and healthy control groups before breakfast, and within 2 h after AMI from the subjects in the AMI group. After centrifugation (Z 366; Hermle Labortechnik GmbH, Germany) at 4°C and 3,000 g for 10 min, the serum was collected and stored in a 1.5-mL RNAse-free EP tube at −80°C. Exosomes were isolated from the serum by ultracentrifugation (XPN-80; Beckman Coulter, United States) at 110,000 g for 80 min at 4°C. The supernatant was discarded, and the precipitate was thoroughly resuspended in 9 mL of phosphate-buffered saline (PBS). The ultracentrifugation was repeated under the same condition, and after discarding the supernatant, 200 μL of PBS was added to the precipitate to prepare an exosome suspension, which was transferred to a 1.5 mL EP tube for preservation.
Xpn 80
Manufactured by Beckman Coulter
Sourced in United States, Germany
The XPN-80 is a centrifugal separator designed for laboratory applications. It is capable of separating various sample types at high speeds to facilitate the isolation of specific components. The XPN-80 features advanced temperature control and programmable operation to ensure consistent and reliable performance.
Automatically generated - may contain errors
Lab products found in correlation
Pestil b, by DWK Life Sciences (4 mentions)
Apex alexa fluor 647 antibody labeling kit protocol, by Thermo Fisher Scientific (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Facsaria 2, by BD (4 mentions)
Macroprep q cartridge, by Bio-Rad (3 mentions)
Complete protease inhibitor cocktail, by Roche (3 mentions)
Type 50.4 ti rotor, by Beckman Coulter (3 mentions)
Alexa fluor 488 conjugated to neun, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by BD (2 mentions)
Alexa fluor 488 conjugated neun, by Merck Group (2 mentions)
Peg 6000, by Merck Group (2 mentions)
Z 366, by Hermle (1 mentions)
Sw41 rotor, by Beckman Coulter (1 mentions)
Thermomixer, by Eppendorf (1 mentions)
Sodium chloride (nacl), by Fresenius (1 mentions)
12 protocols using xpn 80
1
Exosome Isolation from Serum Samples
2
Nuclei Isolation and TDP-43 Profiling
3
TDP-43 and NeuN Positive Nucleus Isolation
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Human autopsy tissue was obtained from the University of Pennsylvania Center for Neurodegenerative Disease Research Brain Bank as described (53 (link)). Informed consent from next of kin was obtained for every case.
Isolation was performed exactly as previously described (52 (link)). Briefly, mid-frontal neocortex of all cases were dounce homogenized using pestil B (Kimble Chase) in 0.25M sucrose and adjusted to final molarity of 1.6M sucrose in TKM. The homogenate was spun on a 1.8M sucrose cushion on the Beckman Coulter XPN-80 ultracentrifuge at 40,000g for 40 minutes at 4°C (Beckman Coulter Inc, Indianapolis, IN, USA). Isolated nuclei were stained with Alexa Fluor 647 conjugated to 2089 (rabbit polyclonal C-terminal anti-TDP-43 antibody, Center for Neurodegenerative Disease Research, University of Pennsylvania), Alexa Fluor 488 conjugated NeuN (EMD Millipore, Billerica, MA, USA), and DAPI (Invitrogen, Carlsbad, CA, USA). Alexa Fluor 647 was conjugated to 2089 according to the APEX Alexa Fluor 647 Antibody labeling kit protocol (Thermo Fisher Scientific, Waltham, MA, USA). Stained nuclei were sorted for single cells containing TDP-43 and NeuN on the BD FACSAria II (BD Biosciences, San Jose, CA, USA) at 20 psi on 100μm nozzle.
Isolation was performed exactly as previously described (52 (link)). Briefly, mid-frontal neocortex of all cases were dounce homogenized using pestil B (Kimble Chase) in 0.25M sucrose and adjusted to final molarity of 1.6M sucrose in TKM. The homogenate was spun on a 1.8M sucrose cushion on the Beckman Coulter XPN-80 ultracentrifuge at 40,000g for 40 minutes at 4°C (Beckman Coulter Inc, Indianapolis, IN, USA). Isolated nuclei were stained with Alexa Fluor 647 conjugated to 2089 (rabbit polyclonal C-terminal anti-TDP-43 antibody, Center for Neurodegenerative Disease Research, University of Pennsylvania), Alexa Fluor 488 conjugated NeuN (EMD Millipore, Billerica, MA, USA), and DAPI (Invitrogen, Carlsbad, CA, USA). Alexa Fluor 647 was conjugated to 2089 according to the APEX Alexa Fluor 647 Antibody labeling kit protocol (Thermo Fisher Scientific, Waltham, MA, USA). Stained nuclei were sorted for single cells containing TDP-43 and NeuN on the BD FACSAria II (BD Biosciences, San Jose, CA, USA) at 20 psi on 100μm nozzle.
4
TDP-43 and NeuN Positive Nucleus Isolation
5
Purification of ADDomers from Insect Cells
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The ADDomers were purified according to a classic protocol.40 (link) Briefly, after lysis of the insect cell pellet by three cycles of freezing-thawing in the presence of Complete protease inhibitor cocktail (Roche) and removal of debris, the lysate was loaded onto a 20%–40% sucrose density gradient. The gradient was centrifuged for 18 h at 4°C at 41,000 rpm on an SW41 rotor in a Beckman XPN-80 ultracentrifuge. The dense collected fractions at the bottom of the tubes were dialyzed against HEPES (10 mM, pH 7.4) and NaCl (150 mM) and then loaded onto a Macroprep Q cartridge (Bio-Rad). After elution by a 150–600 mM linear NaCl gradient in HEPES (10 mM, pH 7.4), ADDomer-containing fractions were checked by SDS-PAGE and concentrated on Amicon (MWCO, 100 kDa) with buffer exchange to HEPES (10 mM, pH 7.4) and NaCl (150 mM).
6
Purification of ADDomers from Insect Cells
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The ADDomers were purified according to classical protocol [15 (link)]. Briefly, after lysis of the insect cell pellet by three cycles of freeze-thaw in the presence of complete protease inhibitor cocktail (Roche, Paris, France) and removal of debris, the lysate was loaded onto a 20% to 40% sucrose density gradient. The gradient was centrifuged for 18 h at 4 °C on an SW 41 Ti rotor in a Beckman XPN-80 ultracentrifuge. The dense collected fractions at the bottom of the tubes were dialyzed against HEPES 10 mM pH 7.4, NaCl 150 mM, and then loaded onto a Macroprep Q cartridge (Bio-Rad, Paris, France). After elution by a 150 to 600 mM linear NaCl gradient in HEPES 10 mM pH 7.4, ADDomer-containing fractions were checked by SDS-PAGE and concentrated on Amicon (MWCO: 100 kDa) with buffer exchange to HEPES 10 mM pH 7.4, NaCl 150 mM.
7
Urine Extracellular Vesicle Isolation
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For the UC-SEC-method, pre-processed urine samples were centrifuged for 45 min at 17,000× g at 4 °C. Next, supernatants were centrifuged for 70 min at 200,000× g and 4 °C in an XPN-80 ultracentrifuge equipped with a Type 50.4 Ti rotor (Beckman Coulter, Krefeld, Germany; k-factor: 51). Obtained pellets were resuspended and washed in 2 mL PBS, followed by pelleting uEVs again by repeating the UC step. SEC of resuspended pellets was performed as described above.
8
Urine Extracellular Vesicle Isolation
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PEG precipitation and subsequent UC was performed as described previously [31 (link)], with some minor modifications. Briefly, pre-processed urine samples were centrifuged for 45 min at 10,000× g and 4 °C. Following addition of 1 mL 50 w/v % PEG-6000 (Sigma-Aldrich, Steinheim, Germany) and 750 µL of 0.9% NaCl (Fresenius Kabi, Bad Homburg, Germany) to 8.25 mL supernatant (final PEG concentration: 10%), uEVs were precipitated at 4 °C overnight. Precipitates were pelleted by centrifugation for 30 min at 1500× g and 4 °C. Pellets were resuspended in 0.9% NaCl and centrifuged at 110,000× g for 2 h at 4 °C in an XPN-80 ultracentrifuge equipped with a Type 50.4 Ti rotor (Beckman Coulter, Krefeld, Germany; k-factor: 93). UC pellets were resuspended in 1 mL of 0.9% NaCl and stored at −80 °C until further analysis.
9
Nuclei Isolation and TDP-43 Profiling
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Middle frontal neocortex was dounce homogenized using pestil B (Kimble Chase, Rockwood, TN, USA) in 0.25M sucrose in TKM (50mM Tris, 25mM KCl, 5 mM MgCl2) buffer. The homogenate was adjusted to 1.6M using 2.3M sucrose in TKM. The homogenate was spun on a 1.8M sucrose cushion in TKM using a SW41 rotor on the Beckman Coulter XPN-80 ultracentrifuge at 40,000 g for 40 minutes at 4 C (Beckman Coulter Inc, Indianapolis, IN, USA). Isolated nuclei were stained with Alexa Fluor 647 conjugated to 2089 (rabbit polyclonal C-terminal anti-TDP-43 antibody, Center for Neurodegenerative Disease Research, University of Pennsylva-nia), Alexa Fluor 488 conjugated to NeuN (EMD Millipore, Billerica, MA, USA), and DAPI (Invitrogen, Carlsbad, CA, USA). Alexa Fluor 647 was conjugated to 2089 according to the APEX Alexa Fluor 647 Antibody labeling kit protocol (Thermo Fisher Scientific, Waltham, MA, USA). Stained nuclei were sorted for single cells based on DAPI, NeuN and TDP-43 fluorescence on the BD FACSAria II (BD Biosciences, San Jose, CA, USA) at 20 psi on 100mm nozzle.
10
Purification of ADDomer and ADD-ST Proteins
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The ADDomer and ADD-ST were purified according to classical protocol.49 (link) Briefly, after lysis of the insect cell pellet by three cycles of freeze-thaw in the presence of Complete protease inhibitor cocktail (Roche), and removal of debris, the lysate was loaded onto a 20% to 40% sucrose density gradient. The gradient was centrifuged for 18 h at 4°C on an SW41 Rotor in a Beckman XPN-80 ultracentrifuge. The dense collected fractions at the bottom of the tubes were dialyzed against Hepes 10 mM pH 7.4, NaCl 150 mM, and then loaded onto a Macroprep Q cartridge (Bio-Rad). After elution by a 150- to 600-mM linear NaCl gradient in Hepes 10 mM pH 7.4, ADDomer-containing fractions were checked by SDS-PAGE and concentrated on Amicon (MWCO: 100 kDa) with buffer exchange to Hepes 10 mM pH 7.4, NaCl 150 mM.
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