After 4 days of 0.5 mM dibutryl-cAMP (Sigma) treatment, F11 cells were treated with 5 nM gp120 (Immunodiagnostics) for 5, 15, or 30 min. An experimental group of cells were first treated with 2 µM AMD3100 for 1 hr at 37℃ prior to treatment with 5 nM gp120 for 5, 15, or 30 min. Treatment with phosphate-buffered saline (PBS), the gp120 diluent, was used as a negative control. After treatment, cells were scraped and collected in 700 μL lysis buffer (1% sodium dodecyl sulfate in PBS, pH 7.4) and sonicated for two 3-sec bursts each. Cells were spun at 14,000 rpm at 4℃ in a Beckman tabletop centrifuge. Protein concentration of clarified lysates was determined using a bicinchoninic acid assay kit (Pierce). Sample buffer was added to the clarified supernatants, and the samples were placed in the −80 ℃ freezer prior to analysis via sodium dodecyl sulfate polyacrylamide gel electrophoresis.
Tabletop centrifuge
Manufactured by Beckman Coulter
The Tabletop Centrifuge is a compact and versatile laboratory equipment designed for separating materials based on their density. It can efficiently separate a variety of samples, including blood, cell cultures, and other liquid mixtures. The centrifuge operates using a motorized rotor to spin the samples at high speeds, creating a centrifugal force that separates the components of the sample based on their different densities.
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Lab products found in correlation
Dibutryl camp, by Merck Group (2 mentions)
Mitotracker green, by Thermo Fisher Scientific (2 mentions)
Annexin 5 pi, by BD (2 mentions)
Mitotracker dye, by Thermo Fisher Scientific (2 mentions)
Annexin 5 binding buffer, by BD (2 mentions)
Lsr 2, by BD (2 mentions)
Sytox blue, by Thermo Fisher Scientific (2 mentions)
Xf96 analyzer, by Agilent Technologies (2 mentions)
Pepstatin a, by Merck Group (1 mentions)
Leupeptin, by Merck Group (1 mentions)
Cm3050 s research cryostat, by Leica (1 mentions)
Aprotinin, by Merck Group (1 mentions)
Calpain inhibitor 1, by Merck Group (1 mentions)
G actin, by Cytoskeleton (1 mentions)
Cholesterol e kit, by Fujifilm (1 mentions)
Beckman ultracentrifuge, by Beckman Coulter (1 mentions)
Pbs solution, by Thermo Fisher Scientific (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
13 protocols using tabletop centrifuge
1
Gp120-Induced Signaling Pathway in F11 Cells
2
Skeletal Muscle Protein Extraction and Analysis
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30 slices of 30 µM thickness were taken with a cryostat (Leica CM3050S Research Cryostat) from skeletal muscle or heart that had been frozen in liquid nitrogen-cooled 2-methylbutane. For biochemical analysis of murine skeletal muscle, quadriceps muscle were used.
Samples were solubilized in 500 µL of 1% Triton X-100 in 50 mM Tris pH 7.6 and 150 mM NaCl with protease inhibitors (per 10 mL buffer: 67 µL each of 0.2 M phenylmethylsulfonylfluoride (PMSF), 0.1 M benzamidine and 5 µL of each of leupeptin (Sigma/Millipore) 5 mg/mL, pepstatin A (Millipore) 1 mg/mL in methanol, aprotinin (Sigma-Aldrich) 5 mg/mL, calpeptin (Fisher/EMD Millipore) 1.92 mg/mL in Dimethyl Sulfoxide (DMSO), Calpain Inhibitor 1 (Sigma-Aldrich) 1.92 mg/mL in DMSO). Samples were vortexed for 4 min and solubilized for 2.5 hr at 4°C with rotation. Samples were then spun down at 12,000 rpm for 30 min at 4°C on a Beckman Tabletop Centrifuge. The supernatant was incubated with 100 µL WGA-Agarose slurry (Vector Biolabs, Malvern, PA, AL-1023) overnight at 4°C with rotation. The next day samples were washed three times in 50 mM Tris pH 7.6 and 150 mM NaCl with 0.1% TX-100 and protease inhibitors. 100 µL of 5X Laemmli Sample Buffer (LSB) was added, samples boiled for 10 min, and 125 µL of this was loaded in each lane of gels for western blotting.
Samples were solubilized in 500 µL of 1% Triton X-100 in 50 mM Tris pH 7.6 and 150 mM NaCl with protease inhibitors (per 10 mL buffer: 67 µL each of 0.2 M phenylmethylsulfonylfluoride (PMSF), 0.1 M benzamidine and 5 µL of each of leupeptin (Sigma/Millipore) 5 mg/mL, pepstatin A (Millipore) 1 mg/mL in methanol, aprotinin (Sigma-Aldrich) 5 mg/mL, calpeptin (Fisher/EMD Millipore) 1.92 mg/mL in Dimethyl Sulfoxide (DMSO), Calpain Inhibitor 1 (Sigma-Aldrich) 1.92 mg/mL in DMSO). Samples were vortexed for 4 min and solubilized for 2.5 hr at 4°C with rotation. Samples were then spun down at 12,000 rpm for 30 min at 4°C on a Beckman Tabletop Centrifuge. The supernatant was incubated with 100 µL WGA-Agarose slurry (Vector Biolabs, Malvern, PA, AL-1023) overnight at 4°C with rotation. The next day samples were washed three times in 50 mM Tris pH 7.6 and 150 mM NaCl with 0.1% TX-100 and protease inhibitors. 100 µL of 5X Laemmli Sample Buffer (LSB) was added, samples boiled for 10 min, and 125 µL of this was loaded in each lane of gels for western blotting.
3
Actin-Based Protein Interaction Assay
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G-actin (rabbit skeletal muscle actin protein, >99% pure; Cytoskeleton, Inc.) was prepared according to the manufacturer’s protocol with G buffer (2 mM Tris, pH 8.0, 0.2 mM CaCl2, 0.2 mM ATP, 0.5 mM DTT). All purified proteins including GST, GST-MT1-MMP-CT, and HIS-MTCBP-1 were dialyzed in G buffer after purification and subjected to centrifugation (100,000 g for 20 min at 4°C) to remove insoluble proteins. The indicated amount of purified proteins and G-actin were added into each individual reaction in a total volume of 200 µl that was equilibrated with G buffer. Subsequently, 20 µl of F buffer (20 mM Tris, pH 7.5, 2 mM CaCl2, 1 M KCl, 20 mM MgCl2, 10 mM ATP, and 5 mM DTT) was added to each reaction and incubated for 1 h before pelleting (100,000 g for 1 h at 25°C) in a Beckman table-top centrifuge. Pellets and supernatants were separated and brought up to equal volume and 40 µl of the samples were resolved by SDS-PAGE gels and immunoblotted.
4
Multiparametric Analysis of Cell Death
5
F11 Cell Differentiation Protocol
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F11 cells were differentiated in 100 mm Petri dishes. After 4 days of 0.5 mM dibutryl-cAMP (Sigma) treatment, cells were scraped and collected in 700 µL lysis buffer (1% sodium dodecyl sulfate [SDS] in phosphate-buffered saline [PBS], pH 7.4) and sonicated for two short, 3-s bursts each. Cells were spun at 14,000 rpm at 4℃ in a Beckman tabletop centrifuge. Protein concentration of the clarified supernatants was determined using a bicinchoninic acid assay (BCA assay) kit (Pierce). Sample buffer was added to the clarified supernatants, and the samples were placed in the −20℃ freezer prior to analysis via SDS-PAGE.
6
Subcellular Fractionation and RNA Extraction
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Cells were spun down in Falcon tubes for 5 min at 3,000 rpm in a Beckman tabletop centrifuge, washed once with PBS, and spun again for 5 min at 3,000 rpm. After completely aspirating the PBS, 800 μl of hypotonic buffer (10 mM Hepes, pH 7.9, 1.5 mM MgCl2, and 10 mM KCl) was added to each sample, the pellet was gently resuspended, transferred to a microfuge tube, and placed in ice for 2 min. Through the remaining steps of the protocol, samples were kept on ice and spun at 4°C. 10% Nonidet P-40 was added to a final concentration of 0.4% (35 μl). Samples were inverted a few times and spun at 3,000 rpm for 7 min. Supernatants (450 μl; cytoplasmic fractions) were collected for processing, and the remaining supernatants were discarded. Then, the pellet (nuclear fraction) was gently resuspended in 500 μl of hypotonic buffer and spun at 3,000 rpm for 2 min. This washing step was repeated three to four times. After removing the buffer following the last spin, the samples were briefly spun once again to remove the remaining supernatant from the cells. Both the cytoplasmic and nuclear fractions were processed following the RNA extraction protocol as recommended by the manufacturer.
7
Measuring Cellular Oxygen Consumption Rates
8
Serum Cytokine and Cholesterol Profiling
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Blood was rested to clot at room temperature for 1 h, and then samples were centrifuged at 1,000g for 10 min at 4 °C in a tabletop centrifuge (Beckman Coulter). The supernatant (serum) was collected and assessed for cytokines and cholesterol content. Total cholesterol analysis was performed using the Wako/Fujifilm Cholesterol-E kit (999-02601), following the manufacturer’s instructions. Multiplex cytokine analysis was performed using LEGENDplex murine inflammatory panel (BioLegend, 740446), following the manufacturer’s protocol and analysis pipeline.
9
Preparation and Use of Porcine PRF
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Fresh blood samples containing 10 mL blood in 10 mL coated glass tubes without anticoagulants were collected from the pig precaval vein. All pigs were female with a mean age of 3.1 months (range from 2.9 to 3.5 months). The mean whole blood platelet count of was 3.9 × 104/μL (3.2 × 104 to 4.66 × 104/μL). All experiments in this study were approved by the Ethics Committee of the University of Illinois at Chicago, IL, USA. As described in previous studies, samples were immediately centrifuged at 2100 rpm (approximately 400 g) for 12 min using a Beckman tabletop centrifuge. The PRF clots were collected between the red corpuscles at the bottom of the centrifuge tube and platelet-poor acellular plasma at the top of the centrifuge tube, and then were gently compressed to form a membrane. For the preparation of lyophilized PRF, PRF membranes were frozen and stored at −80 °C. The frozen PRF was then freeze dried overnight using a Labconco lyophilizer at −51 °C (Free Zone, Labconco, Kansas City, MO, USA). To prepare conditioned medium, either fresh or lyophilized PRF membranes were soaked in 5 mL fresh DMEM medium without fetal bovine serum in 6-well cell culture plates. The conditioned medium was collected every 48 h and fresh medium was added into the wells after collection.
10
Exosome Isolation and Proteinase K Treatment
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The media was harvested from DIV9 E18 rat primary neural cultures and centrifuged at 300 × g for 15 min at 4 °C. The supernatant was collected and centrifuged at 2,000 × g for 15 min at 4 °C in a Beckman tabletop centrifuge. The second supernatant was further centrifuged by using a Beckman ultracentrifuge at 10,000 × g for 45 min at 4 °C. The supernatant was collected and centrifuged at 100,000 × g for 1 h at 4 °C to pellet exosomes. The exosome pellet was resuspended in 1 mL PBS solution and divided equally into 2 tubes. The tubes were incubated at 37 °C for 30 min after 100 µg/mL of Proteinase K was added to one and an equal volume of PBS solution to the other. The exosomes were then washed twice with PBS solution (Gibco) at 120,000 × g for 1 h at 4 °C followed by resuspension in PBS solution to an effective enrichment of 2,400× compared with conditioned culture media. Proteinase K treatment depletes exosomes of proteins (SI Appendix, Fig. S5 ).
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