Livers and spleens were collected at 0 h, 6 h, 24 h and 96 h after P. berghei sporozoite inoculation, mechanically homogenized in PBS containing 2 U/ml DNAse (Sigma-Aldrich), using a 100 μm cell strainer, and centrifuged at 410 x g for 8 min at room temperature (RT). Liver cell pellets were further fractionated using a 35% (v/v) Percoll gradient medium (Sigma-Aldrich) diluted in Roswell Park Memorial Institute 1640 medium (RPMI) (Gibco-Thermo Fisher Scientific, Waltham, MA USA), and centrifuged at 1,360 x g for 20 min without brake at 20°C. Liver and spleen cell pellets were washed with PBS and centrifuged at 410 x g for 8 min at RT. RBCs were depleted by a 3 min incubation at RT with ammonium-chloride-potassium solution, which was stopped by washing with PBS 2% FBS (Gibco-Thermo Fisher Scientific) (FACS buffer). The leukocyte suspension was centrifuged at 410 x g for 8 min at RT and resuspended in PBS for subsequent staining.
Percoll gradient medium
Manufactured by Merck Group
Sourced in United States
Percoll gradient medium is a laboratory product used for the separation and purification of cells, organelles, and other biological particles. It is a colloidal silica solution that forms a density gradient when centrifuged, allowing the separation of different components based on their density.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using percoll gradient medium
1
Isolation of Malarial Liver and Spleen Cells
2
Isolating Mitochondrial-Associated Membranes
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Mitochondrial‐associated membranes (MAM) were isolated according to the protocol from the Wieckowski lab.29 Briefly, to isolate crude mitochondria from HL‐1 cardiomyocytes, homogenized cells were centrifuged twice at 600g for 5 minutes at 4 °C to remove unbroken cells and nuclei. To obtain a mitochondrial‐enriched pellet, the supernatant was collected and centrifuged at 7000g for 10 minutes at 4 °C. To obtain a MAM fraction, the pellet was resuspended and separated in 8 mL Percoll gradient medium (Sigma‐Aldrich) and centrifuged at 950 00g for 30 minutes at 4 °C. The MAM fraction was collected from the Percoll gradient, resuspended, and centrifuged at 100 000g for 1 hour at 4 °C to get the MAM fraction. See Method S1 for detailed information.
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