Percoll

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Percoll is a colloidal silica-based medium used for cell separation and gradient centrifugation. It is designed to provide a density gradient for the isolation and purification of cells, organelles, and other biological particles.

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Percoll gradient (290 citations) Percoll density gradient (39 citations) Percoll gradient medium (4 citations) Percoll reagent (3 citations) Percoll PLUS gradient (3 citations) Isotonic 36% Percoll (2 citations) Percoll buffer (2 citations) Percoll media (2 citations) Percoll® density gradient separation medium (2 citations) Percoll density separation (2 citations)

1 443 protocols using percoll

Percoll Density Gradient Fractionation

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To make a 100% Percoll solution, 9 parts (v/v) of Percoll (GE Healthcare, Piscataway, NJ, USA) was mixed with 1 part (v/v) of 10× concentrated PBS. The 100% Percoll solution was diluted into 5 ml of 40%, 30%, 20%, and 10% Percoll solutions in 1x PBS. Pure 1x PBS was used as 0% Percoll solution. 2 mL of the 40% Percoll gradient solution was layered at the bottom of the 15 ml Falcon tube, and the rest of the 30%, 20%, 10% and 0% Percoll solutions (2 ml each) were added at a 45-degree angle with care not to disturb the layers. The density markers beads (GE Healthcare, Piscataway, NJ, USA) and CSCs and NSTCs incubated at 24 hours at normal media and 1.5 mg/ml Fe(NO₃)₃ were each suspended in cold 20% Percoll solution, added to the top of the Percoll gradient tubes, and centrifuged for 15 minutes at 800 x g. After centrifugation, the visible heights of the cells and the density marker beads were compared for calculation of the density of the cells.

Park K.J., Kim J., Testoff T., Adams J., Poklar M., Zborowski M., Venere M, & Chalmers J.J. (2019). Quantitative Characterization of the Regulation of Iron Metabolism in Glioblastoma Stem-Like Cells using Magnetophoresis. Biotechnology and bioengineering, 116(7), 1644-1655.

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RBC Separation on Percoll Gradient

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For RBC separation on a Percoll (GE Healthcare, Little Chalfont, Buckinghamshire, UK; density 1.13 g/mL) density gradient 0.5 mL of blood sample were gently placed on top of the 90% Percoll mixture [9:1:1.1 of Percoll: 10 × buffer C: 1 × buffer C containing (in mM): 140 NaCl, 4 KCl, 0.75 MgSO₄, 10 glucose, 2 CaCl₂, 0.015 ZnCl₂, 0.2 Gly, 0.2 Glu, 0.2 Ala, 0.1 Arg, 0.6 Gln, 20 HEPES-Imidazole, pH 7.4 at 37°C)]. This buffer was designed taking into accounts recent findings on the role of plasma-borne amino acids and Zn²⁺ ions in control of Ca²⁺ uptake by RBCs (and thereby activity of Gardos channels and RBC volume, Makhro et al., 2013 (link), 2016 (link)) and NO production by endothelial NO synthase in RBCs (Makhro et al., 2010 (link)). Then samples were centrifuged at 45,000 g for 30 min and the distribution of RBCs within the gradient was recorded.

Makhro A., Huisjes R., Verhagen L.P., Mañú-Pereira M.D., Llaudet-Planas E., Petkova-Kirova P., Wang J., Eichler H., Bogdanova A., van Wijk R., Vives-Corrons J.L, & Kaestner L. (2016). Red Cell Properties after Different Modes of Blood Transportation. Frontiers in Physiology, 7, 288.

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Isolation and Characterization of Mouse and Human Liver Sinusoidal Endothelial Cells

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Mouse LSECs were isolated by previously described two-step collagenase perfusion technique with modifications.^{15 (link)} In brief, the liver was perfused with Liver Perfusion Medium (Invitrogen), and dissociated by Liver Digest Medium (Invitrogen). The NPCs were fractionated with Percoll (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) gradient centrifugation with 75% stock Percoll solution and 35% stock Percoll solution. LSEC faction was isolated by mouse LSEC-binding magnetic beads (Miltenyi, Auburn, CA, USA) and Dynabeads Magnetic Beads conjugated with anti-mouse CD31 antibody (MEC13.3, BD Biosciences). Expression of Id1, CXCR7, HGF and Wnt2 messenger RNA was determined. Primary human LSECs were procured from ScienCell Research Laboratories (catalog no. 5000, Carlsbad, CA, USA). Expression of factor VIII was validated by immunostaining. Akt-LSECs were derived from isolated LSECs that were transfected with the pCCL. PGK lentiviral vector with mouse constitutively active Akt1 (myristoylated Akt: myrAkt).^{63 (link)} After starving in serum-free medium, 500 000 LSECs were seeded and stimulated with 10 ng ml⁻¹ SDF-1. LSECs were also treated with 30 μm Wortmannin (Sigma-Aldrich).

Shido K., Chavez D., Cao Z., Ko J.L., Rafii S, & Ding B.S. (2017). Platelets prime hematopoietic–vascular niche to drive angiocrine-mediated liver regeneration. Signal Transduction and Targeted Therapy, 2, 16044-.

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Isolation of Human and Murine Neutrophils

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Blood was collected from the peripheral vein of healthy volunteer donors into heparin tubes, and neutrophils were obtained by first spinning heparinized blood on Ficoll-Paque (Amersham Pharmacia Biotech, Piscataway, NJ) then subjecting the red blood cell (RBC) layer to 1.5% dextran sedimentation, followed by hypotonic lysis as previously described [48 (link)]. Isolated neutrophils were washed with PBS before use. Murine bone marrow-derived neutrophils were isolated, as previously described [49 (link)]. Briefly, femur and tibia were obtained, freed of muscle tissue and flushed with supplemented Hanks’ balanced saline solution (HBSS; 1X HBSS, 0.5% fetal bovine serum (FBS) and 20 mM HEPES) using 10 ml syringe and 30-gauge needle. Cells were pipetted up and down to obtain a single-cell suspension and centrifuged at 300 g for 5 mins. RBCs were lysed as above, and cells were then centrifuged on a discontinuous gradient of 52%, 69%, and 78% Percoll (GE Healthcare) diluted in HBSS (100% Percoll = 9 parts Percoll and 1 part 10X HBSS) and centrifuged (1500 g, 30 mins, without brake). Neutrophils from the 69%/78% interface were collected, washed in PBS, and resuspended in complete RPMI medium for use.

Okeke E.B., Louttit C., Fry C., Najafabadi A.H., Han K., Nemzek J, & Moon J.J. (2020). Inhibition of neutrophil elastase prevents neutrophil extracellular trap formation and rescues mice from endotoxic shock. Biomaterials, 238, 119836.

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Isolation of Lamina Propria Cells

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Lamina propria cells (LP cells) were isolated using a slightly modified previous method [19 ]. Briefly, jejunum tissues were cut into 0.5 cm pieces and washed with phosphate-buffered saline (PBS; Thermo Fisher Scientific, Waltham, MA, USA) containing 1 mM DL-dithiothreitol (DTT; Sigma-Aldrich, St. Louis, MO, USA), 30 mM ethylene-diamine-tetra acetic acid (EDTA; Thermo Fisher Scientific, Waltham, MA, USA), and 10 mM 4-[2-hydroxyethyl]-1-piperazineerhanesulfonic acid (HEPES; Thermo Fisher Scientific, Waltham, WA, USA) at 37 °C for 10 min (predigestion first step). Then, tissue samples were washed again in PBS containing 30 mM EDTA and 10 mM HEPES at 37 °C for 10 min (predigestion second step). After the washing step, tissues were transferred to 5 mL of 10% fetal bovine serum (FBS) containing RPMI 1640 (GenDEPOT, Barker, TX, USA) and inverted for 2 min (neutralization step). Lastly, the tissues were digested in 10% FBS containing RPMI 1640 with 0.5 mg/ml collagenase VIII (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 1 h (digestion step). After the digestion step, isolated cells were applied to Percoll (GE Healthcare, Chicago, IL, USA) gradient centrifugation (40% Percoll on the top, 70% Percoll on the bottom).

Joo S.S., Yoon J.H., Jung J.Y., Joo S.Y., An S.H., Ban B.C., Kong C, & Kim M. (2022). The Modulatory Effects of Lacticaseibacillus paracasei Strain NSMJ56 on Gut Immunity and Microbiome in Early-Age Broiler Chickens. Animals : an Open Access Journal from MDPI, 12(23), 3413.

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Isolating Colonic Lamina Propria Cells

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Colonic lamina propria cells were isolated, as previously described [18 (link)]. Briefly, colon tissues were washed in PBS containing 1 mM DL-dithiothreitol (DTT; Sigma-Aldrich, Irvine, UK), 30 mM ethylenediaminetetraacetic acid (EDTA; Thermo Fisher Scientific/Ambion, Waltham, MA, USA), and 10 mM 4-[2-hydroxyethyl]-1-piperazineerhanesulfonic acid (HEPES; Thermo Fisher, Waltham, MA, USA) at 37 °C for 10 min. The tissues were washed again in PBS containing 30 mM EDTA and 10 mM HEPS at 37 °C for 10 min. After washing, the tissues were digested in RPMI 1640 containing 0.5 mg/mL collagenase VIII (Sigma-Aldrich, Chem, Fort Lauderdale, FL, USA) and 15 ug/mL DNase I (Sigma Aldrich; 90 mg/mL) at 37 °C for 1 h. The cell suspensions from the enzyme digestion were then applied to a Percoll (GE Healthcared/Amersham, Bucking-hampshire, UK) gradient (for lymphocytes: 40% Percoll on the top and 80% Percoll on the bottom) by centrifugation without breaking at room temperature.

Gu B.H., Rim C.Y., Lee S., Kim T.Y., Joo S.S., Lee S.J., Park H.K, & Kim M. (2022). Alteration of Gut Immunity and Microbiome in Mixed Granulocytic Asthma. Biomedicines, 10(11), 2946.

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Isolation of Intestinal Lymphocytes

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LP lymphocytes were isolated as described previously with a simple modification (19 (link)). In brief, the mice were killed, and the colons were removed and opened longitudinally. The intestines were thoroughly washed in PBS and cut into 1.5-cm pieces. The intestines were shaken in PBS containing 1 mM DTT, 30 mM EDTA, and 10 mM Hepes at 37 °C for 10 min. Then the intestines were shaken in PBS containing 30 mM EDTA and 10 mM Hepes at 37 °C for 10 min. After washing with complete RPMI 1640 medium, the tissues were digested in RPMI 1640 containing 16% collagenase VIII (Sigma-Aldrich; 50 KU) and DNase I (Sigma Aldrich; 90 mg/mL) at 37 °C for 55 min. The cell suspensions from the enzyme digestion were then applied to a Percoll (GE Healthcare) gradient (for lymphocytes: 40% Percoll on the top, 80% Percoll on the bottom) by centrifugation at 2500 rpm for 25 min at room temperature. Lymphocytes were harvested from the interphase and washed twice with 0.5% BSA-PBS.

Sun S., Luo L., Liang W., Yin Q., Guo J., Rush A.M., Lv Z., Liang Q., Fischbach M.A., Sonnenburg J.L., Dodd D., Davis M.M, & Wang F. (2020). Bifidobacterium alters the gut microbiota and modulates the functional metabolism of T regulatory cells in the context of immune checkpoint blockade. Proceedings of the National Academy of Sciences of the United States of America, 117(44), 27509-27515.

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Isolation of Human Blood Leukocytes

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Up to 80 mL of whole blood was collected into citrate tubes. An aliquot of 5 mL of whole blood was treated with red cell lysis buffer (Invitrogen) and with the remaining volume, human blood leukocytes were isolated by dextran sedimentation and discontinuous Percoll gradients as described by Dransfield
et al. (2015)
^{23 (link)}. Briefly, blood was first centrifuged at 300 × g (acceleration 5, deceleration 5) for 20 minutes and the platelet-rich plasma layer removed. Erythrocyte sedimentation and leukocyte-rich plasma were obtained by incubating the remaining contents in the tube with 6 mL of 6% Dextran 500 (Pharmacosmos) in saline and final volume adjusted to 50 mL with 0.9% NaCl (Baxter) for at least 20 minutes at room temperature. The leukocyte-rich portion was centrifuged at 350 × g (acceleration 5, deceleration 5) for 6 minutes, with the pellet resuspended in 3 mL of 49.5% Percoll (GE Healthcare) and overlayed onto 61.2% Percoll and 72.9% Percoll. Gradients were centrifuged at 720 × g (acceleration 1, deceleration 0) for 20 minutes to obtain PMN and PBMC layers.

Reyes L., A. Sanchez-Garcia M., Morrison T., Howden A.J., Watts E.R., Arienti S., Sadiku P., Coelho P., Mirchandani A.S., Zhang A., Hope D., Clark S.K., Singleton J., Johnston S., Grecian R., Poon A., McNamara S., Harper I., Fourman M.H., Brenes A.J., Pathak S., Lloyd A., Blanco G.R., von Kriegsheim A., Ghesquiere B., Vermaelen W., Cologna C.T., Dhaliwal K., Hirani N., Dockrell D.H., Whyte M.K., Griffith D., Cantrell D.A, & Walmsley S.R. (2021). A type I IFN, prothrombotic hyperinflammatory neutrophil signature is distinct for COVID-19 ARDS. Wellcome Open Research, 6, 38.

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Isolation of Liver Mononuclear Cells

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Mouse livers were cut into pieces and incubated in collagenase-containing buffer (Type IV, 0.05%) for 30 min followed by pressing with a 200-gauge steel mesh. Then, the cells were collected, suspended with saline, and centrifuged at 1000g for 5 min. The cell pellets were collected and resuspended in 40% Percoll (GE Healthcare, Freiburg, Germany), overlaid gently with 20% Percoll on the 40% Percoll, and then centrifuged for 17 min at 2800g. Liver mononuclear cells were taken from the interphase and centrifuged at 1000g for 5 min before collected as liver mononuclear cells. Hepatic Kupffer cells were isolated as previously described.³¹

Zheng C., Yin S., Yang Y., Yu Y, & Xie X. (2017). CD24 aggravates acute liver injury in autoimmune hepatitis by promoting IFN-γ production by CD4+ T cells. Cellular and Molecular Immunology, 15(3), 260-271.

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Isolation of Mouse Hematopoietic Cells

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BM cell suspensions from all mouse strains described above were prepared by crushing the long bones (2 femurs and 2 tibias per mouse) into DMEM/3% FCS. Bone fragments were removed by filtration through 40-μm filters. Splenocyte suspensions were obtained by mashing the organs through sieves into DMEM/3% FCS, washing by centrifugation and filtering through a 40-μm filter cap. Liver hematopoietic mononuclear cells were obtained by mashing entire livers through sieves, after which the cells were washed in DMEM/3% FCS and centrifuged using a Percoll (GE Healthcare) gradient (40% Percoll layered over 80% Percoll) for 30 mins at 2,000 rpm to remove hepatocytes and other non-hematopoietic cells. Cells localized at the interface were recovered, diluted in DMEM/3% FCS, centrifuged and filtered through 40-μm filter caps. Single-cell suspensions were stained as previously described (20 (link)). Monoclonal antibody conjugates used for flow cytometry are listed in Supplementary Table S1. Cells were analyzed on a 5-laser LSR II cytometer equipped with 355, 405, 488, 561, and 640-nm lasers (Becton Dickinson, San Jose, CA), and the data were analyzed with FlowJo V9 software (TreeStar, Ashland, OR).

Wilson A., Fu H., Schiffrin M., Winkler C., Koufany M., Jouzeau J.Y., Bonnet N., Gilardi F., Renevey F., Luther S.A., Moulin D, & Desvergne B. (2018). Lack of Adipocytes Alters Hematopoiesis in Lipodystrophic Mice. Frontiers in Immunology, 9, 2573.

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