For the induction of a focal cortical stroke, we modified existing models of endothelin-1 (ET-1)-induced brain ischemia42 to avoid traumatic injury to the brain. Under anaesthesia and analgesia (Fentanyl, Midazolam, Medetomidin: 0.05//5//0.5 mg/kg bodyweight), 3-month-old animals were fixed in a stereotactic frame and a circular piece of skull was removed (5 mm diameter, centred on Bregma; as described in43 (link)). The dura mater was carefully removed with the help of a microhook (Fine Science Tools) and 5 µl of ET-1 (Bachem; 64 µM) in Hanks Buffered Salt Solution (Invitrogen) or vehicle solution was topically applied to the cortex and incubated for 10 min. The craniotomy was then covered with a 5 mm glass coverslip, which was fixed in place with dental cement (Hybond), the skin was sutured, then the mice received antidote (Flumazenil, Atipamezol: 0.5//2.5mg/kg bodyweight) and were health-monitored. Control mice underwent the same surgical procedure with application of vehicle solution to the cortex. After 4 weeks, animals were deeply anesthetized and prepared as described above.
Hank s buffered salt solution
Manufactured by Thermo Fisher Scientific
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Hank's Buffered Salt Solution (HBSS) is a balanced salt solution commonly used in cell culture and biological research. It maintains the pH and osmotic environment suitable for maintaining the viability of cells and tissues. HBSS contains inorganic salts, glucose, and a buffering system to help preserve the physiological conditions required for cell growth and experimentation.
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Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (8 mentions)
Trypsin edta, by Thermo Fisher Scientific (8 mentions)
Streptomycin, by Thermo Fisher Scientific (6 mentions)
Dmem f12, by Thermo Fisher Scientific (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Pen strep, by Thermo Fisher Scientific (4 mentions)
Microhook, by Fine Science Tools (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Collagenase 1, by Worthington (3 mentions)
Milli q plus water, by Merck Group (2 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (2 mentions)
Tween 20, by Merck Group (2 mentions)
Diphenyleneiodonium chloride, by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Horse serum, by Thermo Fisher Scientific (2 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
56 protocols using hank s buffered salt solution
1
Focal Cortical Stroke Model via Endothelin-1
2
Assessing mPTP Opening in SK-N-SH Cells
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The opening of the mPTP in cultured SK-N-SH cells was assessed by the Calcein/Co2+-quenching technique as described by using the MitoProbe Transition Pore Assay Kit (Molecular Probes) according to the manufacturer's instructions. Briefly, after treatments, SK-N-SH cells were loaded with 1 µM Calcein-AM (green), 2 mM CoCl2 and 20 nM MitoTracker Red (Invitrogen) at 37°C for 20 min in phenol red free Hank's buffered salt solution (Invitrogen). After washings, live cells were imaged using OLYMPUS FV1000 confocal microscopy with appropriate excitation and emission filters for fluorescein. The mPTP inhibitor cyclosporin A (CsA) (1 µM) (LC laboratories) was applied as a positive control.
3
Cerebellar Slice Culture of SCA17 Mice
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The cerebellar slice culture protocol was modified from a previous report.35 (link) Whole brains were isolated from postnatal day 7 SCA17 mice and transferred to ice-cold culture medium containing 50% basal Medium Eagle (Invitrogen), 25% Hank’s buffered salt solution (Invitrogen), 25% horse serum (Invitrogen), 0.5% D-glucose (Sigma), 1 mM GlutaMax-I, 100 U/mL penicillin, and 100 μg/mL streptomycin. The cerebellum was separated from the other brain regions in ice-cold medium, and the hemisphere was embedded with low-melting-point agarose (Bio Basic) in D-PBS (Invitrogen). The cerebellum was then cut into 350 μm parasagittal sections with a Vibratome (VT1200S; Leica). To improve the survival rate of cerebellar slices, we continuously bubbled the buffer with 95% O2 and 5% CO2 during the sectioning. The slices were then cultured on 0.4 μm pore size culture plate inserts (Millipore) in six-well plates. All treatments were applied to the slices at day 2. After culture for 7 days, cells were immunostained with primary antibodies IP3R-1 and 1TBP18, fluorescence-conjugated secondary antibodies, and 4′,6-diamidino-2-phenylindole, as described earlier. The staining results were observed by confocal microscope.
4
Quantification of Intracellular MTX and its Transporters
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Indican, indoxyl sulfate (IS) and phenolsulfonphthalein (PSP) were obtained from Sigma–Aldrich Chemical Co. (St. Louis, MO, U.S.A.). Methotrexate (MTX) (25.0 mg/mL) was obtained from Wyeth Pharma Gmbh (Wolfratshausen, Germany). MK 571 (purity 98%) was obtained from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). Dimethyl sulfoxide (DMSO), sodium dodecyl sulfate (SDS), 3-(4′,5′-dimethylthiazol-2′-yl)-2,5-diphenyltetrazolium bromide (MTT) and triton X-100 were supplied by Sigma (St. Louis, MO, USA). Fetal Bovine Serum (FBS) was obtained from Biological Industries Inc. (Kibbutz, Beit Haemek, Israel). Penicillin-Streptomycin-Glutamine, Dulbecco’s Modified Eagle Medium (DMEM), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, Hank’s Buffered Salt Solution (HBSS), 5-chloromethylfluorescein diacetate (CMFDA) and trypsin/EDTA were purchased from Invitrogen (Grand Island, NY, USA). TDx kit of MTX was purchased from Abbott Laboratories (Abbott Park, IL, USA). Other reagents were HPLC grade or reagent grade. Milli-Q plus water (Millipore, Bedford, MA, USA) was used throughout this study.
5
Optimizing Cell Culture Conditions
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Dulbecco’s modified Eagle’s medium (DMEM), phosphate-buffered saline (PBS), Hank’s buffered salt solution, Earle’s balanced salt solution, penicillin, streptomycin, human plasma fibronectin, and trypsin-ethylenediaminetetraacetic acid were purchased from Invitrogen Life Technologies, Carlsbad, CA. Type IV collagenase, HEPES (4-[2-hydroxyethyl] piperazine-1-ethanesulfonic acid), glucagon, calcium chloride, hydrocortisone, sodium dodecyl sulfate, hydrogen peroxide, glutaraldehyde, dicumarol, 3-methylcholanthrene, calf thymus DNA, chitosan, and hyaluronic acid (HA) was purchased from Sigma-Aldrich, St. Louis, MO. All other chemicals were purchased from Fisher Scientific, Waltham, MA, unless otherwise noted.
6
Focal Cortical Stroke Model via Endothelin-1
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For the induction of a focal cortical stroke, we modified existing models of endothelin-1 (ET-1)-induced brain ischemia42 to avoid traumatic injury to the brain. Under anaesthesia and analgesia (Fentanyl, Midazolam, Medetomidin: 0.05//5//0.5 mg/kg bodyweight), 3-month-old animals were fixed in a stereotactic frame and a circular piece of skull was removed (5 mm diameter, centred on Bregma; as described in43 (link)). The dura mater was carefully removed with the help of a microhook (Fine Science Tools) and 5 µl of ET-1 (Bachem; 64 µM) in Hanks Buffered Salt Solution (Invitrogen) or vehicle solution was topically applied to the cortex and incubated for 10 min. The craniotomy was then covered with a 5 mm glass coverslip, which was fixed in place with dental cement (Hybond), the skin was sutured, then the mice received antidote (Flumazenil, Atipamezol: 0.5//2.5mg/kg bodyweight) and were health-monitored. Control mice underwent the same surgical procedure with application of vehicle solution to the cortex. After 4 weeks, animals were deeply anesthetized and prepared as described above.
7
Subcutaneous Tumor Xenograft Model
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Animal experiments were approved by the Institutional Review Boards of the Samsung Medical Center and conducted in accordance with the “National Institutes of Health Guide for the Care and Use of Laboratory Animals” (NIH publication 80–23). Nine-week-old male BALB/c nude mice (Orient Bio) were used for subcutaneous injection. 5×106 cells were resuspended in Hank's Buffered Salt Solution (Invitrogen) and mixed with an equal volume of high concentration matrigel (BD Sciences). The mixture was injected into the right dorsal flank of mice. Swelling of the injected lesion over a diameter >200 mm3 was considered as a tumor incidence. Mice with the reduction of the total body weight by >20% were euthanized, and the masses at the injected sites were excised for histological confirmation of the tumors.
8
Induction of Focal Cortical Stroke in Mice
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The induction of focal cortical stroke was performed as described previously (Wendeln et al., 2018 (link)). Briefly, mice were anesthetized with 4% isoflurane in 30% O2 and 70% N2 and maintained on 2% isoflurane in 30% O2 and 70% N2 using a mask. Three-month-old mice were fixed in a stereotactic frame and a circular piece of skull was removed (5 mm in diameter, centered on Bregma). The dura mater was carefully removed with the help of a microhook (Fine Science Tools) and 5 μl of ET-1 (Bachem; 64 μM) in Hanks buffered salt solution (Invitrogen) or vehicle solution was topically applied to the cortex and incubated for 10 min. The craniotomy was then covered with a 5 mm glass coverslip, which was fixed in place with dental cement (Hybond). The skin was sutured and the mice received adrenergic receptor antagonists (flumazenil and atipamezole: 0.5 and 2.5 mg/kg of body weight, respectively) and their health was monitored. Control mice underwent the same surgical procedure with application of vehicle solution to the cortex.
9
Neuronal Regeneration via Functionalized Biomaterials
10
Measurement of Nox-Derived Reactive Oxygen Species
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RPMI 1640 with Glutamax, DMEM/F12 (1:1), Hanks' buffered salt solution (HBSS), fetal bovine serum (FBS), and Amplex red were purchased from Invitrogen, Paisley, UK. Pest (penicillin, streptomycin), neomycin, ionomycin, phorbolmyristateacetate (PMA), diphenyleneiodoniumchloride (DPI), dapsone, ML-171, Phox-I2, xanthine, hypoxanthine, xanthine oxidase, DMSO, DPPH (2,2-diphenyl-1-1picrylhydrazyl), Tween20, Sucrose, flavin adenine dinucleotide (FAD), Phosphatidic acid, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), horseradish peroxidase (HRP) and NADPH were purchased from Sigma–Aldrich. HEK293 overexpressing Nox4 (CJ Nox4) cells were purchased from Redoxis, Lund, Sweden. HEK 293 cells expressing Nox5 and CHO cells expressing Nox1 were a kind gift from the Vincent Jaque Center Medical Universitaire, Geneva, Switzerland. GKT136901, a Nox1/Nox4 selective inhibitor, was a kind gift from prof. Harald HH Schmidt (Maastricht University, Netherlands).
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