Discontinuous percoll gradient

Manufactured by Merck Group

The Discontinuous Percoll Gradient is a laboratory equipment used for the separation and purification of cells, organelles, and other biological particles. It is based on the use of a non-toxic, colloidal silica material called Percoll, which can be used to create density gradients for density-based separation. The Discontinuous Percoll Gradient allows for the efficient isolation and enrichment of specific cell types or subcellular fractions from complex biological samples.

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Lab products found in correlation

Collagenase 4, by Roche (2 mentions) Red blood cell lysis buffer, by BD (1 mentions)

Close spelling variants of this product

Percoll (850 citations) Percoll gradient (157 citations) Discontinuous percoll (3 citations)

5 protocols using discontinuous percoll gradient

Isolation and Characterization of Spinal Cord Leukocytes

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Purification of leukocytes from spinal cords (SC) were performed as described (Fallarino et al., 2010 (link)). Briefly, spinal cords were recovered from anesthetized mice perfused with cold PBS. After centrifugation of spinal cord homogenates, infiltrating leukocytes were separated on a discontinuous percoll gradient (Sigma–Aldrich).
Real-Time PCR (for Rorc, Foxp3, and Ido1) analyses were carried out as described (Fallarino et al., 2010 (link); Pallotta et al., 2011 (link)) and expression of each gene was normalized to Gapdh expression, as determined by the relative quantification method (ΔΔCT) (mean ± SD of triplicate determination).

Volpi C., Mondanelli G., Pallotta M.T., Vacca C., Iacono A., Gargaro M., Albini E., Bianchi R., Belladonna M.L., Celanire S., Mordant C., Heroux M., Royer-Urios I., Schneider M., Vitte P.A., Cacquevel M., Galibert L., Poli S.M., Solari A., Bicciato S., Calvitti M., Antognelli C., Puccetti P., Orabona C., Fallarino F, & Grohmann U. (2016). Allosteric modulation of metabotropic glutamate receptor 4 activates IDO1-dependent, immunoregulatory signaling in dendritic cells. Neuropharmacology, 102, 59-71.

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Isolation and Characterization of Intrahepatic Lymphocytes

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The spleen and thymus were mechanically dissociated. The supernatant was collected and incubated with red blood cell lysis buffer (BD Biosciences) to remove red blood cells. IHLs were isolated according to our previous method^{52 (link)}. Briefly, liver tissue was pressed and digested with collagenase IV (0.05%, Roche Applied Science, Indianapolis, IN) at 37 °C for 30 min. After digestion, cell suspensions were passed through 70-μm nylon cell strainers to yield single-cell suspensions. Cells were purified by centrifugation (400 × g) at room temperature for 30 min over a 30%/70% discontinuous Percoll gradient (Sigma-Aldrich). Then cells were collected from the interphase, thoroughly washed, and re-suspended in complete RPMI-1640 medium containing 10% FBS. The relative percentages of lymphocyte subpopulations were measured by flow cytometry, and their absolute numbers were calculated according to their percentages and the total numbers.

Yi P., Liang Y., Yuan D.M., Jie Z., Kwota Z., Chen Y., Cong Y., Fan X, & Sun J. (2017). A tightly regulated IL-22 response maintains immune functions and homeostasis in systemic viral infection. Scientific Reports, 7, 3857.

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Isolation and Purification of Hippocampal Synaptosomes

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The hippocampal pools of synaptosomes (3 mice per pool) were obtained in accordance with Dunkley et al. (1986)^{43 (link)}. After decapitation and dissection, tissues were immediately homogenized in an ice gradient solution (320 mM sucrose, 0.25 mM dithiothreitol, 1 mM EDTA), centrifuged (1000g, 10 min, 4 °C) and the resultant supernatant was purified by separation in a discontinuous percoll gradient (Sigma Aldrich^®) at concentrations of 23%, 15%, 10%, and 3% (pH 7.4). After centrifugation (6000g, 15 min, 4 °C), the isolated synaptosome was re-suspended in a Krebs–Ringer–HEPES solution (KRH) without CaCl₂ (124 mM NaCl, 4 mM KCl, 1.2 mM MgSO₄, 10 mM glucose, 25 mM HEPES, pH 7.4, 10 mg/ml), and centrifuged (6000g, 15 min, 4 °C). The Pellet was obtained, centrifuged (3.333g, 60 s), re-suspended in KRH, and incubated at 37 °C per 30 min. This process was repeated once more and synaptosome aliquots were immediately led for reading and quantification.

Ribeiro R., Silva E.G., Moreira F.C., Gomes G.F., Cussat G.R., Silva B.S., da Silva M.C., de Barros Fernandes H., de Sena Oliveira C., de Oliveira Guarnieri L., Lopes V., Ferreira C.N., de Faria A.M., Maioli T.U., Ribeiro F.M., de Miranda A.S., Moraes G.S., de Oliveira A.C, & Vieira L.B. (2023). Chronic hyperpalatable diet induces impairment of hippocampal-dependent memories and alters glutamatergic and fractalkine axis signaling. Scientific Reports, 13, 16358.

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Spinal Cord Leukocyte Purification and Gene Expression

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Purification of leukocytes from spinal cords (SC) were performed as described (Fallarino et al., 2010 (link)). Briefly, spinal cords were recovered from anesthetized mice perfused with cold PBS. After centrifugation of spinal cord homogenates, infiltrating leukocytes were separated on a discontinuous percoll gradient (Sigma–Aldrich).
Real-Time PCR (for Rorc, Foxp3, and Ido1) analyses were carried out as described (Fallarino et al., 2010 (link), Pallotta et al., 2011 (link)) and expression of each gene was normalized to Gapdh expression, as determined by the relative quantification method (ΔΔCT) (mean ± SD of triplicate determination).

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Isolation and Purification of Mouse Intrahepatic Lymphocytes

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Mouse IHLs were isolated, according to our previous method with slight modifications (32 (link)). In brief, the inferior vena cava was cut above the diaphragm, and the liver was perfused with cold PBS through the portal vein. Liver tissues were collected and digested in RPMI 1640 containing collagenase IV (0.05%; Roche Applied Science, Indianapolis, IN) at 37°C for 30 min. After digestion, cell suspensions were passed through 70-μm nylon cell strainers to remove aggregates and yield single-cell suspensions. IHLs were purified by centrifugation (400 g) at room temperature for 30 min over a 30/70% discontinuous Percoll gradient (Sigma-Aldrich St. Louis, MO). The IHLs were collected from the interphase, thoroughly washed, and resuspended in complete RPMI 1640 containing 10% FBS (Hyclone, Logan, UT). The absolute numbers of IHLs per liver were counted by a light microscopy. The relative percentages of cell populations were analyzed by flow cytometry, and the absolute numbers of these lymphocyte subpopulations were calculated by their percentages and IHL numbers.

Wang X., Liang Y., Wang H., Zhang B., Soong L., Cai J., Yi P., Fan X, & Sun J. (2022). The Protective Role of IL-36/IL-36R Signal in Con A-Induced Acute Hepatitis. Journal of immunology (Baltimore, Md. : 1950), 208(4), 861-869.

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