The nodose ganglia were excised from anesthetized rats and placed in ice-cold Ca2+- and Mg2+-free Hank’s balanced salt solution (Gibco, Grand Island, NY, USA) supplemented with penicillin and streptomycin (100 U/mL and 100 μg/mL, respectively; Gibco). The ganglia were cleaned of connective tissue and desheathed, and then were enzymatically dissociated by incubation in HEPES-buffered DMEM/F-12 (DMEM/F-12; Gibco) containing collagenase Ia (1 mg/mL; Sigma-Aldrich), dispase II (1 mg/mL; Sigma-Aldrich), and DNase I (100 U/mL; Sigma-Aldrich) at 37°C for 40 minutes. Thereafter, they were mechanically dissociated by passing 5–10 times through a fire-polished glass Pasteur pipette before separation with a Percoll PLUS (Sigma-Aldrich) density gradient. Individual cells were harvested by centrifugation at 700 × g for 3 minutes. They were then plated onto coverslips precoated with poly-L-lysine (Sigma-Aldrich) and were incubated overnight at 37°C in DMEM/F-12 with 10% fetal bovine serum (Gibco) and antibiotics in a humidified 5% CO2 atmosphere.
Percoll plus
Manufactured by Merck Group
Sourced in United States
Percoll PLUS is a sterile, ready-to-use density gradient medium for the separation and purification of cells, organelles, and other biological particles. It is composed of colloidal silica particles coated with polyvinylpyrrolidone, which provides a stable, iso-osmotic environment for the separation process.
Automatically generated - may contain errors
Lab products found in correlation
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Dispase 2, by Merck Group (1 mentions)
Mg2 free hank s balanced salt solution, by Thermo Fisher Scientific (1 mentions)
Collagenase ia, by Merck Group (1 mentions)
Ficoll paque premium 1, by GE Healthcare (1 mentions)
May grunwald stain, by Merck Group (1 mentions)
Fcr blocking reagent, by Miltenyi Biotec (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
L glutamine, by Lonza (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
6 protocols using percoll plus
1
Isolation and Culture of Rat Nodose Ganglion Neurons
2
Monocyte isolation via Ficoll-Paque and Percoll
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Monocytes were isolated as previously described, using Ficoll-Paque Premium 1.073 (GE Healthcare, Uppsala, Sweden) [12 (link)] and hyperosmotic Percoll Plus (Sigma-Aldrich, St. Louis, MO, USA; prepared as described by Repnick et al. [13 (link)]) or by discontinuous density centrifugation using isotonic Percoll Plus (Sigma-Aldrich, St. Louis, MO, USA) at densities 1.070 g mL−1, 1.062 g mL−1, 1.060 g mL−1 and 1.058 g mL−1. Monocytes within a band located at the interface of the Percoll solution and DPBS/media were collected and diluted with 4 mL of Ca2+/Mg2+ free DPBS containing 1 mmol L−1 EDTA. An aliquot of the cells was stained with May-Grunwald stain (Merck Millipore, Burlington, MA, USA) to confirm the presence of monocytes [14 (link)].
3
Purification of Cells using Percoll PLUS
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A working solution of Percoll PLUS (Sigma Aldrich), with initial density of 1.130 g ml−1, was prepared by diluting Percoll to 72% in 0.15 M NaCl. Self-generating gradients of cells in Percoll were prepared by mixing 7.2 ml of Percoll working solution and 0.8 ml cell suspension in 0.15 m NaCl and centrifugation at 23,000×g at 20 °C for 30 min in a fixed-angle rotor (A27 8 × 50) in a Sorvall RC6+ centrifuge. The density of the self-generated gradients was determined by measuring the refractive index with a digital refractometer (ORD 1RS, Kern Optics, Germany) of control samples without cells, taken from the tubes at different distances from the bottom and using a standard curve (refractive index vs density) supplied by the manufacturer.
4
Isolation and Treatment of Neutrophils
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Citrated whole blood was obtained by venepuncture and red blood cells removed by dextran sedimentation for 45 minutes. Neutrophils were isolated from the cell-enriched plasma by Percoll PLUS (Sigma, UK) gradient centrifugation at 700 g for 30 minutes and resuspended in phenol-free RPMI (Thermo Scientific, UK) supplemented with 10% FCS (Thermo Scientific, UK) and 2mM L-glutamine (Lonza, Switzerland). Isolated neutrophils were counted using a 0.4% Trypan Blue solution and diluted to 2 × 106 neutrophils/ml to ensure the same number of viable cells were used between groups and experiments. For experiments examining the effects of IgG, cells were treated with a FcR blocking reagent (Miltenyi, UK) (2 μl per 1 × 106 neutrophils) for 20 minutes prior to addition of 200 μg/ml purified IgG.
5
Microalgae cultures for colloid tracking
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Cultures of CR strain CC125 were grown axenically in a Tris-Acetate-Phosphate medium32 at 21 °C under continuous fluorescent illumination (100 μE m−2 s−1, OSRAM Fluora). Cells were harvested at ∼5 × 106cells ml−1 in the exponentially growing phase, then centrifuged at 800 r.p.m. for 10 min and the supernatants replaced by DI-water (sedimentation experiment) or by a Percoll solution (tracking and spreading experiments; Percoll Plus, Sigma) already containing the desired concentration of PS colloids (Polybead Microspheres, diameter d=1±0.02 μm).
6
Cryopreserved Sperm Purification and Analysis
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Cryopreserved straws were thawed using a heating pad at 37 °C for 30 s. Then, the whole volume of each straw was transferred into labeled 0.5 mL tubes, and the proportion of motile spermatozoa was evaluated with the computer-assisted sperm analysis (CASA) system. After that, the samples were washed three times by adding 500 µL of PBS (phosphate saline solution; Sigma-Aldrich, St. Louis, MO, USA) and centrifuged for 10 min/5000 RPM for the disposal of egg yolk extender residues.
For the extraction of RNA and proteins, the cell suspensions were subjected to a single-layer Percoll® Plus (Sigma-Aldrich, St. Louis, MO, USA) density gradient separation, according to Ďuračka et al. [85 (link)].
For the extraction of RNA and proteins, the cell suspensions were subjected to a single-layer Percoll® Plus (Sigma-Aldrich, St. Louis, MO, USA) density gradient separation, according to Ďuračka et al. [85 (link)].
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