The blood from individual animals was collected 12 h after SCI into EDTA tubes to obtain plasma. Frozen samples of isolated spinal cord were homogenized (Dounce homogenizer, Thomas Scientific, Swedesboro, NJ, USA) and transferred to 10 volumes (w/w) of ice-cold PBS supplemented with 0.1% Tween 20 and a protease inhibitor cocktail. The prepared spinal cord homogenate was vortexed for 15 s and subjected to rotator for 5 min at room temperature. Further cell debris was removed by centrifugations at 4 °C (30,000× g for 10 min). The supernatant was further used for measurement. Milliplex Rat Cytokine/Chemokine Magnetic Bead Panel (Merck, Rahway, NJ, USA) was used to measure the levels of chemokines and cytokines in plasma and spinal cord extracts. Samples were diluted at 1:4 ratio and further incubated with magnetic beads, washed up, and then incubated with detecting antibodies and SA-PE. The data were obtained using a Luminex 200 analyzer using xPONENT software (Luminex, Austin, TX, USA).
Dounce homogenizer
Manufactured by Thomas Scientific
Sourced in United States
The Dounce homogenizer is a laboratory equipment used for the mechanical disruption of cells and tissues. It consists of a glass or Teflon pestle that fits snugly inside a matching glass or Teflon cylinder, allowing for the gentle and controlled homogenization of samples.
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Lab products found in correlation
Invitrogen human aβ42 elisa kit, by Thermo Fisher Scientific (2 mentions)
A32965, by Thermo Fisher Scientific (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Sw41 rotor, by Beckman Coulter (2 mentions)
Macs smartstrainer, by Miltenyi Biotec (1 mentions)
Total rna purification kit, by Jena Biosciences (1 mentions)
Script cdna synthesis kit, by Jena Biosciences (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Ne per nuclear and cytoplasmic extraction kit, by Thermo Fisher Scientific (1 mentions)
Ne per nuclear and cytoplasmic extraction reagent kit, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Chemidoc gel imaging system, by Bio-Rad (1 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (1 mentions)
Atp fluorometric assay kit, by Abcam (1 mentions)
15 protocols using dounce homogenizer
1
Cytokine and Chemokine Profiling after SCI
2
Total RNA Purification and cDNA Synthesis
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Total RNA purification kit, SCRIPT cDNA Synthesis kit and qPCR GreenMaster with UNG/lowROX – blue dyed as well as primers were all obtained from Jena Bioscience (Jena Germany). Materials for tissue culture which include Gentamycin (10 mg/ml) antibiotics, Dulbecco's modified Eagle's medium and fetal bovine serum (FBS) were obtained from the WHO Polio Laboratory, Department of Virology, University of Ibadan. Hep-2 cell line used in the study was obtained from Infectious Disease Unit of Luxembourg Institute of Health. Fluorochrome-conjugated antibodies used for immunophenotyping analysis were produced by Biolegend, eBiosciences, BD Biosciences and Beckon Dickinson Co., and donated freely by Prof. Ross M. Kedl of the Department of Immunology and Microbiology, University of Colorado Denver, Aurora Colorado. 70 μM wire mesh MACS Smart Strainer was a product of Miltenyi Biotec GmbH (Bergisch Gladbach, Germany) while a hand-operated 7 ml Dounce Homogenizer used in the study was manufactured by Thomas Scientific, Swedesboro NJ, USA. Unless stated otherwise, other standard chemicals and reagents were obtained from Sigma-Aldrich (St. Louis, MO).
3
Proteasome Fractionation and MBP Binding
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The 20S and 26S proteasomes from BALB/c mouse brains were separated by ultracentrifugation. The tissue was homogenized (Dounce homogenizer, Thomas Scientific) in 3 V buffer containing 20 mM Tris-HCl (pH 7.5), 10% glycerol, 150 mM NaCl, 1 mM EDTA, 5 mM MgCl2, 1 mM DTT, and protease inhibitor cocktail (Roche), and the homogenates were centrifuged (16,000 g, 4°C 30 min) to remove cell debris. To study MBP binding to the proteasome, the homogenates were incubated with purified bovine MBP in the presence of 1 μM PS-341 for 30 min at 4°C. Further homogenates were separated by ultracentrifugation in 10% to 55% glycerol gradient in buffer containing 20 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 1 mM DTT, and 1 mM ATP at 125,000 g for 18 h at 4°C. The proteasome activity in the resulting fractions was measured using Suc-LLVY-MCA as a substrate in the presence and absence of 1 μM PS-341 and 0.02% SDS.
4
Mitochondrial Isolation from ARPE-19 Cells
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ARPE-19 cells (107 cells/well) were washed in Tris-based Mg2+/Ca2+-deficient buffer (135 mM NaCl, 5 mM KCl, and 25 mM Tris, pH 7.6) and were allowed to swell for 10 min in ice-cold hypotonic CaRSB buffer (10 mM NaCl, 1.5 mM CaCl2, 10 mM Tris, pH 7.5, and 1x protease inhibitor cocktail). The cells were disrupted by 60 strokes in a Dounce homogenizer (Thomas Scientific, Swedesboro, NJ, USA), and mitochondria stabilization buffer (210 mM mannitol, 70 mM sucrose, 5 mM EDTA, and 5 mM Tris, pH 7.6) was added to stabilize the mitochondria. After collecting the nuclei by centrifuging the cells twice at 3000 r.p.m. for 15 min, the supernatant was spun at 14 000 r.p.m. for 20 min at 4 °C. The pellet and the supernatant included the mitochondrial and cytoplasmic fractions, respectively. Cells (5 × 107) were swollen in ice-cold hypotonic CaRSB buffer and were disrupted using a Dounce homogenizer. Cell homogenates were then spun down twice at 3000 r.p.m. for 15 min.
5
Quantifying Human Aβ42 in 5XFAD Mouse Cortex
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Cortical tissue was harvested at 49 weeks of age, and prepared according to the protocol established for 5XFAD mice by Oakley, et al.10 (link). Specifically, following extraction, the tissue was flash frozen and re-suspended in 3 to 4 volumes of PBS-0.5% Triton supplemented with protease inhibitors (A32965 ThermoFisher, Waltham, MA). Tissue was homogenized with a Corning Dounce homogenizer (1234F35 Thomas Scientific, Swedesboro, NJ), freeze–thawed 3 times, and cleared at 15,000 rpm for 15 min at 4 °C. Cleared homogenates were supplemented with guanidine hydrochloride to a final concentration of 5 M to solubilize plaques. To detect human Aβ42 levels, cleared homogenates were diluted in Standard Diluent Buffer and measured by the Invitrogen Human Aβ42 ELISA kit (KHB3441, ThermoFisher, Waltham, MA) following manufacturer’s instructions. Final guanidine hydrochloride concentrations were under 0.1 M. All samples were run in duplicate; their readings fell within the range of the standard dose responses curve. Total protein was measured by a protein assay kit (5000002, Bio-Rad, Hercules, CA) and used for normalization.
6
Nuclear and Cytosolic Fractionation Protocol
7
Protein Extraction and Western Blot
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Livers were homogenized in a Dounce homogenizer (Thomas Scientific) with 30 strokes in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM Ethylenediaminetetraacetic acid (EDTA)) with complete protease and phosphatase inhibitor cocktail. Lysates were sonicated, centrifuged, and boiled in 4× loading buffer. The samples were separated by Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Polyvinylidene fluoride (PVDF) membranes, blocked in 5% nonfat milk, and incubated with the indicated primary antibodies overnight. Secondary antibodies were added for another 1 h. and detected with Clarity Western ECL Substrate (Biorad). Immunoreactive bands were exposed in an automatic X-ray film processor. Antibodies are listed in SI Appendix, Table S1 .
8
Viral Titer Determination by Plaque Assay
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Each sample was serially diluted tenfold and incubated on Vero cells on 12-well plates in duplicate. The inoculum was removed after 1 h of incubation and the cells were overlaid with 1% (w/v) methylcellulose in DMEM containing 10% (w/v) FBS. Six days post-infection, the cells were fixed with 2% (w/v) crystal violet in 20% (v/v) ethanol. Viral titers were determined by counting plaque numbers. To determine viral titers in the mouse tissues, 1-mL homogenates were prepared in a Dounce homogenizer (Thomas Scientific, Swedesboro, NJ, USA) and used for the plaque assay. Plaques were counted and viral titers in each tissue were expressed in PFU mL−1.
9
Protein Extraction and Analysis
10
Membrane Fractions Isolation and Analysis
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The above membrane fractions were lysed in 2 mL of 2-(N-morpholino) ethanesulfonic acid (MES)-buffered saline (MBS: 25 mM MES, 150 mM NaCl, pH 6.0) with protease inhibitors (PIs; Roche Applied Science, Penzberg, Germany) and homogenized with a pre-chilled Dounce homogenizer (Thomas Scientific, Swedesboro, NJ, USA) with 20 strokes. Samples were centrifuged at 1,000 × g for 5 min. The supernatant (2 mL) was adjusted to contain 45% sucrose by adding 2 mL of 90% sucrose in MBS. Following this, 4 mL 35% sucrose in MBS/Na2CO3 (250 mM) and 4 mL 5% sucrose MBS/Na2CO3 were, in turn, layered on top of the supernatant. After centrifugation at 39,000 RPM for 16–10 h in an SW41 rotor (Beckman Instruments, Brea, CA, USA), a light-scattering band at the 5%–35% sucrose interface was collected. Twelve (1 mL) fractions were collected, starting from the top of the gradient. For immunoblotting, an equal amount of total protein from each fraction (25 mg) was analyzed.
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