The brain microvessels were extracted from the brains of 5xFAD mice utilizing a previously described method [19 (link)]. The brains were homogenized in ice-cold DPBS followed by the addition of one volume of 30% Ficoll 400 (Sigma-Aldrich, St. Louis, MO, USA) to a final concentration of 15%; following this, the mixture was mixed, and then centrifuged at 8000× g for 10 min. The resulting pellets were suspended in ice-cold DPBS containing 1% and passed over a glass bead (Kimble Chase, LLC; Vineland, NJ, USA) column to collect microvessels. The microvessels adhering to the glass beads were collected by gentle agitation in 1% BSA in DPBS. The collected microvessels were lysed with a RIPA buffer containing a complete mammalian protease inhibitor (Sigma-Aldrich, MO, USA) and used for an analysis by Western blot.
Ficoll 400
Manufactured by Merck Group
Sourced in United States, Germany, Switzerland
Ficoll 400 is a synthetic, highly branched, and neutral polymer of sucrose. It is commonly used as a density gradient medium for the separation and isolation of cells, organelles, and other biological particles during centrifugation.
Automatically generated - may contain errors
Lab products found in correlation
Ficoll 70, by Merck Group (11 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Glutamax, by Thermo Fisher Scientific (3 mentions)
Analytical grade salts, by Merck Group (2 mentions)
Sucrose, by Merck Group (2 mentions)
Dextran 40, by Merck Group (2 mentions)
Nocodazole, by Merck Group (2 mentions)
Glutathione sepharose 4 fast flow, by GE Healthcare (2 mentions)
Protein a sepharose cl 4b, by GE Healthcare (2 mentions)
Protease inhibitor mixture tablets complete edta free, by Roche (2 mentions)
Puromycin, by Merck Group (2 mentions)
Restriction enzyme, by New England Biolabs (2 mentions)
Collagenase p, by Roche (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Trypan blue, by Merck Group (2 mentions)
Staphylococcus aureus protein a, by Merck Group (2 mentions)
Poly l lysine (pll), by Merck Group (2 mentions)
76 protocols using ficoll 400
1
Brain Microvessels Isolation Protocol
2
Tau-EGFP Liquid-Like Droplet Formation
3
Rat Brain Isolation and Characterization
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Ethylene glycol tetraacetic acid (EGTA), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), glucose, sucrose, Ficoll 400, and analytical grade salts were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acridine orange and Rhodamine 6G were obtained from Molecular Probes (Eugene, OR, USA) and L-[14C]glutamate and aqueous counting scintillant (ACS) from Amersham (Little Chalfont, UK).
Wistar male rats, 100-120 g body weight, were obtained from the vivarium of MD Strazhesko Institute of Cardiology, Medical Academy of Sciences of Ukraine. Animals were kept in animal facilities of the Palladin Institute of Biochemistry in accordance with the European guidelines and international laws and policies (34 ). They were housed in a quiet, temperature-controlled room (22-23°C) and were given water and dry food pellets ad libitum. Rats were decapitated and the brain was removed. Experimental protocols were approved by the Animal Care and Use Committee of the Palladin Institute of Biochemistry (Protocol from September 19, 2012).
Wistar male rats, 100-120 g body weight, were obtained from the vivarium of MD Strazhesko Institute of Cardiology, Medical Academy of Sciences of Ukraine. Animals were kept in animal facilities of the Palladin Institute of Biochemistry in accordance with the European guidelines and international laws and policies (34 ). They were housed in a quiet, temperature-controlled room (22-23°C) and were given water and dry food pellets ad libitum. Rats were decapitated and the brain was removed. Experimental protocols were approved by the Animal Care and Use Committee of the Palladin Institute of Biochemistry (Protocol from September 19, 2012).
4
Amyloid fibril formation inhibition
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The crowding agents, Ficoll 70 and Ficoll 400, were purchased from Sigma-Aldrich (St. Louis, MO). ThT was also obtained from Sigma-Aldrich. Guanidine hydrochloride (GdnHCl) was obtained from Promega (Madison, WI). Proteinase K and Triton X-100 were purchased from Ameresco (Solon, OH). All other chemicals used were made in China and were of analytical grade.
5
Isolation and Purification of Synaptosomes
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Synaptosomes were isolated from forebrains as described 66 . All procedures were carried out at 4°C with pre-cooled reagents and equipment. All buffers were supplemented with protease and phosphatase inhibitors. Brains were homogenized in 1ml sucrose buffer (in mM: 320 mM sucrose, 1 NaHCO3, 1 MgCl2, 0.5 CaCl2)/30mg tissue in a glass Dounce homogenizer with 12 strokes on ice. The homogenate was centrifuged at 2,500rpm for 10min to remove cell debris and nuclei. Pellet was re-homogenized in sucrose buffer and spun again. Supernatants were combined and cleared from any residual debris by spinning at 1,200rpm for 10min. Cleared supernatants were subsequently centrifuged at 9,000rpm to obtain cytosolic supernatant and a pellet containing crude synaptosomes. Pellet was resuspended in 400μl sucrose buffer, pipetted onto 1.8ml 5% Ficoll-400 (Sigma Aldrich), and spun for 45min at 19,300rpm. The supernatant was removed and pellet was re-homogenized in 400μl 5% Ficoll-400. The homogenate was layered on 1.8ml 13% Ficoll-400 and again spun for 45min at 19,300rpm. Synaptosomes form a layer at the interface of 5% and 13% Ficoll-400. The layer was carefully removed and diluted to 1ml with 2% Ficoll-400.Then, synaptosomes were pelleted by centrifugation at 32,000rpm for 30min, after which they were taken up in 100μl 5% SDS, followed by sonication for 10sec at 20% amplitude.
6
Isolation and Purification of Synaptosomes
7
Isolation and Culture of Aortic Cells
8
Boar Semen Sperm Selection
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The boar semen
was provided by Varkens KI Nederland BV. A Ficoll-based separation
technique was used for selection of good-quality spermatozoa. The
semen was centrifuged (300g, 20 min) over a density
gradient of Ficoll-400 (Sigma-Aldrich, Zwijndrecht, The Netherlands),
which separates cells by their motility. The discontinuous density
gradient of Ficoll-400 was obtained with a 40% (v/v) density top layer
and 80% (v/v) density lower layer. The motile spermatozoa formed a
soft pellet at the bottom of the tube, collected, and then dispersed
in the Ringer rinsing solution. After the second centrifugation (500g, 10 min), the pellet was dispersed in noncapacitating
(modified Human Tubal Fluid, mHTF, without BSA, NaHCO3,
and CaCl2)33 (link) or capacitating
medium (Human Tubal Fluid, HTF–NaCl 101.6 mM, KCl 4.69 mM,
glucose 2.78 mM, KH2PO4 0.37 mM, MgSO4 0.2 mM, sodium lactate 21.4 mM, sodium pyruvate 0.33 mM, BSA 4 mg/mL,
NaHCO3 25 mM, CaCl2 2.04 mM).34 (link) Sperm cells were counted in a Bürker-Türk
chamber under the microscope. The capacitation status was confirmed
as described in the Supporting Information (seeFigure S3 ).
was provided by Varkens KI Nederland BV. A Ficoll-based separation
technique was used for selection of good-quality spermatozoa. The
semen was centrifuged (300g, 20 min) over a density
gradient of Ficoll-400 (Sigma-Aldrich, Zwijndrecht, The Netherlands),
which separates cells by their motility. The discontinuous density
gradient of Ficoll-400 was obtained with a 40% (v/v) density top layer
and 80% (v/v) density lower layer. The motile spermatozoa formed a
soft pellet at the bottom of the tube, collected, and then dispersed
in the Ringer rinsing solution. After the second centrifugation (500g, 10 min), the pellet was dispersed in noncapacitating
(modified Human Tubal Fluid, mHTF, without BSA, NaHCO3,
and CaCl2)33 (link) or capacitating
medium (Human Tubal Fluid, HTF–NaCl 101.6 mM, KCl 4.69 mM,
glucose 2.78 mM, KH2PO4 0.37 mM, MgSO4 0.2 mM, sodium lactate 21.4 mM, sodium pyruvate 0.33 mM, BSA 4 mg/mL,
NaHCO3 25 mM, CaCl2 2.04 mM).34 (link) Sperm cells were counted in a Bürker-Türk
chamber under the microscope. The capacitation status was confirmed
as described in the Supporting Information (see
9
Isolation and Characterization of Brain Microvessels
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Brain microvessels were isolated as described previously [69 (link)]. Brain hemispheres were homogenized in ice-cold DPBS followed by the addition of one volume of 30% Ficoll 400 (Sigma-Aldrich). Homogenate was centrifuged at 8000× g for 10 min, and the resulting pellet was suspended in ice-cold DPBS containing 1% BSA and passed over a glass bead column to collect microvessels adhering to the glass beads. Isolated microvessels were used to determine the expression of tight junction proteins ZO-1 and claudin-5, and Aβ transport proteins P-gp, BCRP, RAGE, and LRP1 by Western blot.
10
Fluorescence Anisotropy Protocol for Protein Binding
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The probes used were as follows: /5RhoR-XN/-TCTTCGGAGGGCTGTCACCCGAATATA-3′ (IDT) and its complementary strand (does not contain the fluorophore). The DNA was annealed with 1 μM each strand in water. Fluorescence anisotropy was conducted with the Fluorolog from Horiba with temperature control and fluorescence anisotropy modules in a semi-micro quartz cuvette with light path 10 × 4 mm (Hellma. #114F-10-40). For measurement, DNA was diluted to 2 nM in 800 μL renaturation buffer containing 20 mM HEPES (pH = 7.5), 50 mM NaCl, 50 μg/mL BSA (EMD Millipore, #2960-500GM) and 5% Ficoll 400 (Sigma-Aldrich, #F2637-5G), then titrated with protein starting at 6.2 nM until the anisotropy values are saturated. For binding measurement in dark, the sample was kept in dark for 10 min before anisotropy measurement at each titration. For binding measurement in blue light, the sample was irradiated with 450 nm blue LEDs at an intensity of 1.9 mW/cm2 for 10 min before anisotropy measurement at each titration.
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