Total cerebellum tissue was collected at necropsy from animal J02370 in R10 media (RPMI 1640 + 10% FBS, 2 mM L-glutamine, and 1% PenStrep). Tissue was transferred to a biosafety cabinet where it was rinsed with medium and minced in a tissue culture dish with isolation medium (DMEM/F12 + 10% FBS, 2 mM L-glutamine, and 1% PenStrep). Minced tissue was washed with PBS and further processed by trypsin-mediated enzymatic digestion (DMEM + 4.5 g/L glucose + 0.25% trypsin + 3 U/mL Dornase) at 37°C for 30 min followed by vigorously pipetting to create a homogeneous mix. The trypsinization was stopped by addition of DMEM with 20% FBS. The trypsin mixture was filtered with a 100-μm nylon membrane and the filtrate was pelleted using a tabletop centrifuge spinning for 10 min at 1,200 rpm. The supernatant was aspirated, the pellet was resuspended in a 30% Percoll/PBS solution (GE Healthcare Life Sciences), and centrifuged for 1.5 h at 4,700 rpm with no brake. The microglia-enriched phase, which migrated between myelin debris and blood vessel phases, was extracted using a syringe, filtered through a 40-μm mesh to remove small capillaries, and washed with isolation medium. The cells were pelleted by centrifugation at 2,000 rpm for 10 min, resuspended, and processed for immunostaining and flow cytometry.
Percoll pbs solution
Manufactured by GE Healthcare
Sourced in United Kingdom
Percoll/PBS solution is a laboratory reagent used for cell separation and purification. It is a colloidal silica suspension in phosphate-buffered saline (PBS) that can be used to create density gradients for the isolation of specific cell types from complex mixtures such as blood, bone marrow, or other tissues. The solution allows for the separation of cells based on their density, size, and other physical properties.
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Lab products found in correlation
2 protocols using percoll pbs solution
1
Isolation of Microglial Cells from Cerebellum
2
Isolation and Purification of Merozoites from Babesia bovis Culture
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The method described by Ishizaki et al. (2016) (link) was used to isolate free merozoites liberated from infected RBCs. A five ml sample of an in vitro culture of B. bovis with 30% parasitemia was incubated on ice for 2 h. The culture was then resuspended in five ml of GIT medium. The suspension was slowly overlaid onto two ml of 30% (1.043 g/ml density) Percoll/PBS solution (GE Healthcare, Buckinghamshire, UK) at the bottom of a 15 ml centrifuge tube (Corning, Corning, NY, USA). The tube was centrifuged at 280×g for 5 min and then at 330×g for 20 min at 4 °C. The medium along with the free merozoite layers were transferred carefully to a new tube and then centrifuged at 1,500×g for 5 min at 4 °C. The pellets containing free merozoites were washed twice with 20 ml of GIT medium and then suspended in one ml of GIT medium. A concentration of purified merozoites was calculated with a disposable hemo-cytometer (AR Brown, Tokyo, Japan). The viability of the merozoites was determined after they were stained with 6-carboxyfluorescein diacetate (6-CFDA; Invitrogen Corp., Carlsbad, CA, USA) and propidium iodide (PI; Dojindo, Kumamoto, Japan).
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