Isotonic percoll

Manufactured by GE Healthcare

Sourced in Sweden, United States, United Kingdom, France

Isotonic Percoll is a laboratory equipment used for the separation and isolation of cells and organelles through density gradient centrifugation. It is a colloidal silica solution that can be used to create density gradients for the isolation of a variety of cell types, including but not limited to, lymphocytes, granulocytes, and platelets. The product is designed to maintain the isotonicity of the samples during the separation process.

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53 protocols using isotonic percoll

Purifying proximal SI LP cells

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Isolated LP cells from the proximal SI were further purified by Percoll density gradient centrifugation. Cells were resuspended in 40% isotonic Percoll (GE Healthcare, diluted with PBS) and carefully laid on top of 75% isotonic Percoll. After centrifugation for 20 min at 800 g (without brake), cells were collected from the interface between the 40% and 75% Percoll layers. The cells were washed twice with cold Roswell Park Memorial Institute (RPMI) media and then cultured at a density of 4 × 10⁵ cells in 125 μL of modified complete RPMI media containing 10% FBS and a variable amount of amino acids for 48 h at 37 °C with 5% CO₂. The RPMI media were prepared with either 1, 2, 4, 6, 8, or 12 times the normal amount of amino acids provided in standard Minimum Essential Medium (MEM) media (Gibco). Live cells (exhibiting weak staining with Zombie Yellow dye) were quantified by flow cytometry before and after culture, and cell viability after 48 h was calculated as a percentage of the initial input.

I. Kutyavin V., Korn L.L, & Medzhitov R. (2024). Nutrient-derived signals regulate eosinophil adaptation to the small intestine. Proceedings of the National Academy of Sciences of the United States of America, 121(5), e2316446121.

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Isolation of CNS Mononuclear Cells

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Mononuclear cells were isolated from the CNS as previously described ⁴² with minor modifications. Naïve and EAE mice were euthanatized, and then subjected to perfusion through the left ventricle with ice-cold sterile PBS. Brains and spinal cord were then removed, minced and enzymatically dissociated with 0.05% (w/v) collagenase type III (Worthington Biochemical), 0.5% Dispase II (Roche Applied Science), 40 µg/ml DNAse I, 20 mM HEPES in HBSS) for 30 min at 37C to make a suspension of single cells. Enzymes were inactivated with 20 ml of Ca²⁺/Mg²⁺-free HBSS containing 2 mM EDTA and 20 mM HEPES. The digested tissue was triturated and passed through a 100-µM cell strainer. Cells were centrifuged and resuspended in 30% isotonic Percoll (GE Healthcare) and 40 µg/ml DNAse I, underlined by 70% isotonic Percoll, and centrifuged at 1000 × g at 4°C for 25 min. Cells were collected from the 70%–30% interphase, and sorted by FACS Aria.

Mayo L., Trauger S.A., Blain M., Nadeau M., Patel B., Alvarez J.I., Mascanfroni I.D., Yeste A., Kivisäkk P., Kallas K., Ellezam B., Bakshi R., Prat A., Antel J.P., Weiner H.L, & Quintana F.J. (2014). B4GALT6 regulates astrocyte activation during CNS inflammation. Nature medicine, 20(10), 1147-1156.

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Isolation of Thymic and Splenic Cells

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Thymus and spleen were dissected into RPMI (ThermoFisher Scientific) containing 5% newborn calf serum (NCS) (Atlanta Biologicals) and mechanically teased with forceps to prepare a single-cell suspension. Splenocytes were treated with ACK Lysing Buffer (Lonza) for 3 min on ice to eliminate red blood cells. When analyzing newborn and adult thymic epithelial cells, single-cell thymic suspensions were enzymatically digested with Liberase TM (63 μg/ml; Roche) and DNase I (20 μg/ml; Roche) for 40 min shaking at 37 °C. Following digestion, epithelial cells were enriched by centrifugation in a Percoll gradient. Briefly, cells were resuspended in 4 ml of 1.115 g ml⁻¹ isotonic Percoll (GE Healthcare Life Science) and overlaid with 2 ml of 1.065 g/mL isotonic Percoll, followed by a layer of 2 ml of PBS. Samples were then centrifuged at 2700 RPM at 4 °C for 30 min with the brakes off. The TEC enriched samples were taken from the interface between the Percoll and PBS layer. Embryonic thymi were disaggregated in 0.25% trypsin/0.02% EDTA (Sigma) solution at 37 °C for 7–10 min, with a single-cell suspension made by gentle repetitive pipetting. Embryonic thymi were not subject to an enrichment step. All cell suspensions were then further processed and stained as described below.

Cowan J.E., Malin J., Zhao Y., Seedhom M.O., Harly C., Ohigashi I., Kelly M., Takahama Y., Yewdell J.W., Cam M, & Bhandoola A. (2019). Myc controls a distinct transcriptional program in fetal thymic epithelial cells that determines thymus growth. Nature Communications, 10, 5498.

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Isolation of Mouse Choroid Plexus and Brain Tissue

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Spleens and brains were harvested from mice after transcardial perfusion with ice cold HBSS (Cellgro, Manassas, VA, USA). Single cell suspensions of spleens were prepared by mechanical disruption, and residual red blood cells were lysed using ACK lysis buffer (Quality Biologicals, Gaithersburg, MD, USA) for 5 min at room temperature. The CP was isolated from the brain by careful dissection and the tissue was dissociated in 0.25% trypsin–2.21 mM EDTA (Cellgro) for 30 min at 37°C. Cells were washed twice with ice cold HBSS supplemented with 2% heat inactivated fetal bovine serum (GIBCO, Auckland, New Zealand) and then used for downstream applications.
Brain tissue devoid of CP [ex-choroid plexus (ex-CP)] was dissociated in a digestion buffer containing Liberase TL (3.25 U/ml; Sigma, St. Louis, MO, USA), DNase I (0.1 mg/ml; Sigma), and BSA (1%; Sigma) in HBSS (with Ca²⁺ and Mg²⁺; GIBCO) for 30 min at 37°C. EDTA (1 mM; Sigma) was added to the solution and the cell suspension was filtered through a 40 μm filter (BD, San Diego, CA, USA) and centrifuged at 1,500 rpm for 15 min at 4°C. Isotonic Percoll (30%) (GE Healthcare, Uppsala, Sweden) was added to the pellet, and the suspension carefully layered onto 70% of Isotonic Percoll. The gradient was centrifuged for 30 min at 20°C and the cells at the 70–30% interphase were collected, washed, and used for downstream applications.

Jain S., Stock A., Macian F, & Putterman C. (2018). A Distinct T Follicular Helper Cell Subset Infiltrates the Brain in Murine Neuropsychiatric Lupus. Frontiers in Immunology, 9, 487.

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Isolation and Purification of Immune Cells

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Whole blood, lymph node, and bone marrow samples were processed as previously described (40 (link)). Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by density gradient centrifugation using Ficoll-Paque PLUS (Cytiva) and centrifuged at 1,860 x g for 30 minutes. Plasma was saved for viral nucleic acid and Leronlimab concentration detection. Buffy coat containing PBMCs was collected and washed with R10 (RPMI 1640 containing 10% fetal bovine serum (FBS)) before use. Lymph nodes were collected in R10, diced with a scalpel, and forced through a 70 μm cell strainer to a single cell suspension in R10. Bone marrow aspirates were collected in R10 and pelleted by centrifugation at 830 x g for 4 minutes. Cell pellets were resuspended by shaking in PBS containing 2 mM EDTA, and then centrifuged at 830 x g for 4 minutes. Cell pellets were then resuspended in 70% isotonic Percoll (GE Healthcare, Buckinghamshire, UK) and layered under 37% isotonic Percoll. Layers were centrifuged at 500 x g for 20 minutes. Mononuclear cells in the interface were collected into R10.

Chang X.L., Wu H.L., Webb G.M., Tiwary M., Hughes C., Reed J.S., Hwang J., Waytashek C., Boyle C., Pessoa C., Sylwester A.W., Morrow D., Belica K., Fischer M., Kelly S., Pourhassan N., Bochart R.M., Smedley J., Recknor C.P., Hansen S.G, & Sacha J.B. (2021). CCR5 Receptor Occupancy Analysis Reveals Increased Peripheral Blood CCR5+CD4+ T Cells Following Treatment With the Anti-CCR5 Antibody Leronlimab. Frontiers in Immunology, 12, 794638.

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Isolation and Analysis of CNS-Infiltrating Immune Cells

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Following procedures as previously described (12 (link)), mice were perfused intracardially with ice-cold PBS immediately after sacrifice to remove blood from the intracranial vessels. The brain and spinal cord were prepared with sterile instruments, minced with a scalpel, and incubated with agitation in RPMI 1640 (PAA) containing collagenase A (1 mg ml⁻¹) (Roche) and recombinant ribonuclease-free deoxyribonuclease I (0.1 mg ml⁻¹) (Roche) for 45 min at 37°C. Tissue was triturated through a 100-μm cell strainer and washed with PBS (pellet was centrifuged at 300g for 10 min at 4°C). The homogenized tissue was resuspended in 30% isotonic Percoll (GE Healthcare) and carefully underlaid with 78% isotonic Percoll. After gradient centrifugation (1500g for 30 min at 4°C), CNS-infiltrating immune cells were recovered from the gradient interphase and washed in ice-cold PBS. Single-cell suspensions were stained in the presence of 123count eBeads (Thermo Fisher Scientific). The antibodies and the respective antigen, host species, supplier, catalog number, clone, and dilution are listed in table S3. Data were acquired on an LSR II FACS analyzer (BD Biosciences). Data analysis was performed with the FlowJo v.10 analysis software (FlowJo LLC).

Rothammer N., Woo M.S., Bauer S., Binkle-Ladisch L., Di Liberto G., Egervari K., Wagner I., Haferkamp U., Pless O., Merkler D., Engler J.B, & Friese M.A. (2022). G9a dictates neuronal vulnerability to inflammatory stress via transcriptional control of ferroptosis. Science Advances, 8(31), eabm5500.

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Brain Immune Cell Isolation and Analysis

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Mice were deeply anesthetized with ketamine/xylazine prior to cardiac perfusion with ice cold PBS. Brains were removed aseptically, collected in 7 ml FACS buffer (PBS + 2% FBS), and homogenized using a dounce homogenizer. After the addition of 3 ml of 100% isotonic Percoll (GE Healthcare), the homogenate was underlayed with 1 ml of 70% isotonic Percoll. After centrifugation at 2470 rpm for 30 min at 4°C, cells at the interphase were washed in FACS buffer prior to antibody staining. Anti-coagulated blood was incubated with PharmLyse buffer (BD Biosciences) for 2 minutes on ice according to the manufacturer's protocol. UV live/dead cell stain kit (Invitrogen) was used to assess cell viability, and cells were blocked for non-specific Fc-mediated interactions with 0.5 ug purified anti-mouse CD16/CD32 (BD Biosciences) for 20 minutes at 4°C prior to antibody staining. The following antibodies were used: CD3-FITC (17A2), CD8-PE (53.6.7), CD4-PerCp5.5 (GK1.5), Ly6C-FITC (HK1.4), Ly6G-PE (1A8), CD11b-APC (M1/70), CD45-APC-Cy7 (30-F11), NK1.1-PE (PK136) and CD19-APC (eBio1D3) (all from eBioscience). Data were collected using the LSR II flow cytometer (Becton-Dickinson) and analyzed using FlowJo software 8.5.3 (Treestar). Cell numbers were quantified using counting beads (Spherotech).

Michlmayr D., Bardina S.V., Rodriguez C.A., Pletnev A.G, & Lim J.K. (2016). Dual function of Ccr5 during Langat virus encephalitis - Reduction of neutrophil-mediated CNS inflammation and increase in T cell-mediated viral clearance. Journal of immunology (Baltimore, Md. : 1950), 196(11), 4622-4631.

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Enrichment of Microglia and Astrocytes

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At 14 h after the last cycle of RSD, microglia and astrocytes were isolated from whole brain homogenates as previously reported (Norden et al., 2014 (link)). In brief, brains were homogenized in phosphate-buffered saline (PBS, pH 7.4) by passing through a 70 μm cell strainer. The resulting homogenates were centrifuged at 900 × g for 6 min. Supernatants were removed and cell pellets were re-suspended in 70% isotonic Percoll (GE-Healthcare). A discontinuous Percoll (GE-Healthcare) density gradient (50%, 35%, and 0%) was overlaid and centrifuged at 2000 × g for 20 min. Enriched microglia were collected from the interphase between the 70% and 50% Percoll layers. These cells were characterized as enriched brain CD11b⁺ cells, as previous studies have demonstrated that viable cells isolated by Percoll density gradient yields >90% CD11b⁺ cells (Wohleb et al., 2011 (link), Wohleb et al., 2013 (link)). Enriched astrocytes were collected from the interphase between the 50% and 35% Percoll layers. As previously reported (Norden et al., 2014 (link)), 65–70% of the cells collected from this interphase were characterized as GLAST-1⁺ astrocytes.

Sawicki C.M., McKim D.B., Wohleb E.S., Jarrett B.L., Reader B.F., Norden D.M., Godbout J.P, & Sheridan J.F. (2014). Social defeat promotes a reactive endothelium in a brain region-dependent manner with increased expression of key adhesion molecules, selectins and chemokines associated with the recruitment of myeloid cells to the brain. Neuroscience, 302, 151-164.

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Isolation of CD11b+ Brain Cells

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CD11b⁺ cells were isolated from whole brain homogenates as previously described (McKim, Weber et al. 2017 (link)). In brief, brains and spinal cords were passed through a 70-μm nylon cell strainer and centrifuged at 600 × g for 6 min. Supernatants were removed and cell pellets were re-suspended in 70% isotonic Percoll (GE-Healthcare). A discontinuous Percoll density gradient was layered as follows: 50, 35, and 0% isotonic Percoll. The gradient was centrifuged for 20 min at 2000 × g and cells were collected from the interphase between the 70 and 50% Percoll layers. These cells were referred to as enriched CD11b⁺ cells based on previous studies demonstrating that viable cells isolated by Percoll density gradient yields >90% CD11b⁺ cells (Wohleb, Powell et al. 2013 (link)) (n = 6 per group).

Sawicki C.M., Kim J.K., Weber M.D., Jarrett B.L., Godbout J.P., Sheridan J.F, & Humeidan M. (2017). Ropivacaine and Bupivacaine Prevent Increased Pain Sensitivity Without Altering Neuroimmune Activation Following Repeated Social Defeat Stress. Brain, behavior, and immunity, 69, 113-123.

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Isolation of Brain Leukocytes and Microglia

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Microglia and brain-infiltrating leukocytes were enriched using a modified protocol described previously (McKim et al., 2018 (link)). Mice were euthanized 5 h (early time point for leukocyte infiltration), 15 h (peak leukocyte infiltration time point (Ching et al., 2005 (link))) or 24 h (time point for microglial analysis) after ICV injections and perfused transcardially with cold 1 × PBS (pH 7.4) to flush circulating leukocytes away from the brain. Brains were dissected, homogenized with Potter-Elvehjem Tissue Grinders (Wheaton) and centrifuged at 900 × g for 6 min. Supernatants were removed and cell pellets were re-suspended in 35% isotonic Percoll (GE-Healthcare) and centrifuged for 20 min at 2000 × g. Supernatants were removed and cell pellets were re-suspended in 1 × PBS for cell surface antigen labeling.

Liu X., Nemeth D.P., McKim D.B., Zhu L., DiSabato D.J., Berdysz O., Gorantla G., Oliver B., Witcher K.G., Wang Y., Negray C.E., Vegesna R.S., Sheridan J.F., Godbout J.P., Robson M.J., Blakely R.D., Popovich P.G., Bilbo S.D, & Quan N. (2019). Cell-Type-Specific Interleukin 1Receptor 1 Signaling in the Brain Regulates Distinct Neuroimmune Activities. Immunity, 50(2), 317-333.e6.

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