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Percoll

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Percoll is a colloidal silica-based medium used for density gradient centrifugation to isolate and purify cells, organelles, and other biomolecules. It is an inert, non-toxic, and non-ionic material that can form density gradients with a wide range of densities. Percoll is commonly used in various applications, including cell separation, organelle isolation, and protein purification.

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Lab products found in correlation

Dnase 1, by Merck Group (3 mentions) Collagenase 8, by Merck Group (2 mentions) Dispase, by Merck Group (2 mentions) Ethylene diamine tetraacetic acid (edta), by Promega (2 mentions) Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (2 mentions) Collagenase d, by Roche (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) B27 supplement, by Thermo Fisher Scientific (2 mentions) Mhcii, by BioLegend (1 mentions) Cd107a, by BioLegend (1 mentions) Collagenase a, by Merck Group (1 mentions) Zombie nir, by BioLegend (1 mentions) Cd11b, by BioLegend (1 mentions) F4 80, by BioLegend (1 mentions) Cd11c, by BioLegend (1 mentions) Collagenase, by Worthington (1 mentions) Bovine fibronectin, by Merck Group (1 mentions) Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (1 mentions) Rat tail collagen type 1, by Merck Group (1 mentions) Hanks balanced salt solution (hbss), by Avantor (1 mentions)

10 protocols using percoll

1

Single-cell RNA-seq of Peritoneal Inflammation

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Mice C57BL/6(J) underwent surgery to receive injury alone (PB) or in combination with bacterial contamination (PB + CLP). After 24 h mice were euthanized by injection anesthesia and subsequent intracardial perfusion with ice cold PBS with 2 mM EDTA. Peritoneal buttons were excised and digested for 30 min at 37 °C in IMDM (Gibco, #12440061) containing Ca, 25 mM Hepes, 2% FCS, 1 mg/ml Collagenase 1a (Sigma, #C9891-1g) and 0.1 mg/ml DNAseI (Roche, #10104159001). IMDM with 2 mM EDTA was used to stop the reaction and wash the cells. Then, samples were resuspended in 40% Percoll (VWR, #17-0891-01) and pipetted on top of 80% Percoll solution to create a gradient. After centrifugation (650 × g, 20 min), the interface was transferred again and washed in DPBS + 0.04% BSA. Finally, the sample was resuspended in DPBS + 0.04% BSA for library preparation. RNA-seq libraries were prepared from 10000 cells using the Chromium Single Cell 3’ Library & Gel Bead Kit v3 (10xGenomics, #PN-1000075). Libraries were prepared according to the manufacturer’s protocol. Sequencing was performed on a NovaSeq 6000 S2 flow cell operated by NovaSeq Control Software (v. 1.7). The function cellranger count from Cell Ranger was used to transform the fastq files. The reference genome was the mm10 available at Illumina Cell Ranger webpage. Next, we used the function cellranger mat2csv to generate the UMI matrix.
Zindel J., Mittner J., Bayer J., April-Monn S.L., Kohler A., Nusse Y., Dosch M., Büchi I., Sanchez-Taltavull D., Dawson H., Gomez de Agüero M., Asahina K., Kubes P., Macpherson A.J., Stroka D, & Candinas D. (2021). Intraperitoneal microbial contamination drives post-surgical peritoneal adhesions by mesothelial EGFR-signaling. Nature Communications, 12, 7316.
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2

Isolation and Characterization of Brain Leukocytes

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Leukocytes were isolated from whole brains after digestion in 0.05% collagenase A (Sigma-Aldrich, #C0130) and 10 mg/ml DNase I (Sigma-Aldrich, #D4527), then purified via centrifugation in 37% isotonic Percoll (VWR, # 89428-524) as described [38 (link)]. Isolated leukocytes from brains were stained as described [39 (link)] with fluorescently conjugated antibodies to CD3e (Biolegend, clone 17A2), CD44 (Biolegend, clone IM7), CD19 (Biolegend, clone 6D5), CD107a (Biolegend, clone 1D4B), CD8a (Biolegend, clone 53-6.7), CD4 (Biolegend, clone RM4-5), CD45.2 (Biolegend, clone 104), MHC-II (Biolegend, clone M5/114.15.2), NK1.1 (Biolegend, clone PK136), CD11c (Biolegend, clone N418), F4/80 (Biolegend, clone BM8), CD11b (Biolegend, clone M1/70), Ly6G (Biolegend, clone 1A8), Ly6C (Biolegend, clone HK1.4), CD80 (Biolegend, clone 16-10A1), and Zombie NIR (Biolegend, 423105). Data collection and analysis were performed using an Cytek Northern Lights Cytometer and FlowJo software (Treestar).
Kung P.L., Chou T.W., Lindman M., Chang N.P., Estevez I., Buckley B.D., Atkins C, & Daniels B.P. (2022). Zika virus-induced TNF-α signaling dysregulates expression of neurologic genes associated with psychiatric disorders. Journal of Neuroinflammation, 19, 100.
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3

Isolation of Primary Mouse Endothelial Cells

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The isolation of primary mouse endothelial cells was carried out according to Duport and colleagues [59 (link)]. Briefly, newborn C57BL/6 wildtype mice (4d) were decapitated, and the cortex was subsequently isolated on an ice-cold surface. The tissue was chopped and digested in 0.1% Collagenase (Worthington, Lakewood, NJ, USA) at 4 °C overnight. To isolate endothelial cells from the suspension, a centrifugation step with Percoll (VWR, Darmstadt, Germany) followed. Endothelial cells were counted and seeded in collagen-coated cell culture dishes (Sarstedt, Nümbrecht, Germany) for further expansion in an endothelial cell medium. For an artificial blood-brain barrier, endothelial cells were seeded on collagen-coated membranous cell culture inserts for 6-well-plates (Millipore, Burlington, MA, USA) at a density of 1 × 105 cells/well.
Ruiz-Perera L.M., Höving A.L., Schmidt K.E., Cenan S., Wohllebe M., Greiner J.F., Kaltschmidt C., Simon M., Knabbe C, & Kaltschmidt B. (2021). Neuroprotection Mediated by Human Blood Plasma in Mouse Hippocampal Slice Cultures and in Oxidatively Stressed Human Neurons. International Journal of Molecular Sciences, 22(17), 9567.
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4

Isolation of Colonic Lamina Propria Lymphocytes

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Lamina propria lymphocytes were isolated with minimal modifications to previous published work55. In brief, colons were harvested and fileted, then washed with 1 mL PBS to remove stool before resting in complete RPMI media. After rinsing with 1X HBSS (without Ca2+ and Mg2+), colons were then each incubated, with shaking, in 15 mL 1X HBSS (without Ca2+ and Mg2+) supplemented with 5 mM EDTA (Promega) and 1 mM DL-Dithiothreitol (DTT) (Bioplus chemicals) for 45 min at 37°C in order to remove mucus. Samples were then filtered with a 100 µM sterile cell strainer to discard supernatant before tissue digestion for 45 min at 37°C in 15 mL each of 1X HBSS (with Ca2+ and Mg2+) supplemented with 5% (v/v) fetal bovine serum (GIBCO heat inactivated), 1 U/mL Dispase (Sigma), 0.5 mg/mL Collagenase VIII (Sigma), and 20 µg/mL DNaseI (Sigma). After quenching with 15 mL of complete RPMI media, the supernatant was filtered over a 40 µM sterile cell strainer and collected in a new tube. Cell suspensions were pelleted 1,000 g for 10 min at 4°C, then subjected to a Percoll (VWR) gradient (40%/80% [v/v]) before pelleting at 1,000 g for 20 min with no brake and no acceleration. Cells at the interface were collected and washed with PBS.
Merana G.R., Dwyer L.R., Dhariwala M.O., Weckel A., Gonzalez J.R., Okoro J.N., Cohen J.N., Tamaki C.M., Han J., Tasoff P., Palacios-Calderon Y., Ha C.W., Lynch S.V., Segre J.A., Kong H.H., Kattah M.G., Ma A, & Scharschmidt T.C. (2022). Intestinal inflammation alters the antigen-specific immune response to a skin commensal. Cell reports, 39(9), 110891.
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5

Granulocyte Isolation and Activation Assay

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Percoll was obtained from VWR (Mississauga, ON, Canada) and density gradients were prepared in Dulbecco’s phosphate buffered saline (DPBS) and HBSS at a concentration of 65% (v/v). Recombinant human eotaxin-1 (CCL11) was obtained from Reprokine (Tel Aviv, Israel). NH4Cl lysis buffer used during granulocyte isolation contained (in mM): 155 NH4Cl, 10 KCO3 and 0.1 ethylenediaminetetraacetic acid (EDTA), pH 7.2. All reagents were dissolved in RPMI 1640 Media, which sometimes contained 10% FBS and penicillin/streptomycin (1000 IU) Fisher Scientific, Walkersville, MD, USA), or in (Ca2+-free) HBSS (Gibco, Grand Island, NY, USA), pH 7.2–7.4. DPBS, McCoy’s 5A Medium and fluo-3 AM were obtained from Invitrogen (Burlington, ON, Canada). CDP-323 (Zaurategrast) was obtained from Adooq Bioscience (Irvine, CA, USA). Recombinant Arg-Gly-Asp (RGD) tripeptide, rat tail collagen (Type I), and bovine fibronectin were obtained from Sigma Aldrich (Oakville, ON, Canada). MACS buffer used during eosinophil enrichment was prepared in 1× DPBS and contained the following (in millimoles): 2 EDTA and 0.5% BSA (w/v).
Ahmadzai M., Small M., Sehmi R., Gauvreau G, & Janssen L.J. (2015). Integrins are Mechanosensors That Modulate Human Eosinophil Activation. Frontiers in Immunology, 6, 525.
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6

Isolation of Liver Lymphocytes and Bone Marrow Cells

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Mouse livers and bone marrow were harvested and prepared into single cell suspensions.35 (link) Briefly, to isolate liver lymphocytes, the tissues were physically dissociated using a glass tissue dounce homogenizer and purified using of 40% Percoll (VWR). After centrifugation at 2000rpm for 5 minutes at room temperature, the pellets were resuspended in 1X PBS and filtered using 100μm strainers. To harvest bone marrow, the tibia, fibula, and femur were removed and then crushed using mortar and pestle in 1X PBS. The resulting mixture was filtered using 100μm strainers and centrifuged. Red blood cells in the liver and bone marrow were lysed using ACK lysis buffer. The resulting pellets were either plated for bone marrow cDCP generation or analyzed via flow cytometry.
Hildreth A.D., Padilla E.T., Tafti R.Y., Legala A.R, & O’Sullivan T.E. (2023). Sterile liver injury induces a protective tissue-resident cDC1-ILC1 circuit through cDC1-intrinsic cGAS-STING-dependent IL-12 production. Cell reports, 42(2), 112141.
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7

Isolation and Stimulation of Lymphocytes

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LNs were digested 30 min in IMDM medium (Invitrogen, Zug, Switzerland) supplemented with 1 mg/ml collagenase D (Roche, Rotkreuz, Switzerland). Single cell suspensions were obtained by gently disrupting LNs through a 70 μm strainer, followed by washing with HBSS. Lungs were perfused with 10 ml PBS and digested 45 min and processed as described above. Afterwards, a 30% Percoll (VWR, Nyon, Switzerland) gradient was applied to the cells to isolate lung leukocytes.
For T cell restimulation followed by intracellular staining and flow cytometry, cells were incubated for 6 h at 37 °C in the presence of 100 μg/ml OVA protein with the addition of 5 μg/ml Brefeldin A (Sigma-Aldrich) for the last 3 h of culture. All cells were cultured in IMDM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) (all from Invitrogen). For T cell restimulation followed by ELISA, cells were incubated for 3d at 37 °C in the presence of 100 μg/ml OVA protein in IMDM medium supplemented with 10% FBS and 1% P/S (all from Invitrogen).
Ballester M., Jeanbart L., de Titta A., Nembrini C., Marsland B.J., Hubbell J.A, & Swartz M.A. (2015). Nanoparticle conjugation enhances the immunomodulatory effects of intranasally delivered CpG in house dust mite-allergic mice. Scientific Reports, 5, 14274.
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8

Isolation of Colonic Lamina Propria Lymphocytes

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Lamina propria lymphocytes were isolated with minimal modifications to previous published work55. In brief, colons were harvested and fileted, then washed with 1 mL PBS to remove stool before resting in complete RPMI media. After rinsing with 1X HBSS (without Ca2+ and Mg2+), colons were then each incubated, with shaking, in 15 mL 1X HBSS (without Ca2+ and Mg2+) supplemented with 5 mM EDTA (Promega) and 1 mM DL-Dithiothreitol (DTT) (Bioplus chemicals) for 45 min at 37°C in order to remove mucus. Samples were then filtered with a 100 µM sterile cell strainer to discard supernatant before tissue digestion for 45 min at 37°C in 15 mL each of 1X HBSS (with Ca2+ and Mg2+) supplemented with 5% (v/v) fetal bovine serum (GIBCO heat inactivated), 1 U/mL Dispase (Sigma), 0.5 mg/mL Collagenase VIII (Sigma), and 20 µg/mL DNaseI (Sigma). After quenching with 15 mL of complete RPMI media, the supernatant was filtered over a 40 µM sterile cell strainer and collected in a new tube. Cell suspensions were pelleted 1,000 g for 10 min at 4°C, then subjected to a Percoll (VWR) gradient (40%/80% [v/v]) before pelleting at 1,000 g for 20 min with no brake and no acceleration. Cells at the interface were collected and washed with PBS.
Merana G.R., Dwyer L.R., Dhariwala M.O., Weckel A., Gonzalez J.R., Okoro J.N., Cohen J.N., Tamaki C.M., Han J., Tasoff P., Palacios-Calderon Y., Ha C.W., Lynch S.V., Segre J.A., Kong H.H., Kattah M.G., Ma A, & Scharschmidt T.C. (2022). Intestinal inflammation alters the antigen-specific immune response to a skin commensal. Cell reports, 39(9), 110891.
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9

Isolation of Lamina Propria Cells from Intestine

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Colon and small intestine were removed from mesenteric fat tissue, opened longitudinally and subsequently cleaned in cold phosphate-buffered saline (PBS) and Hank’s Balanced Salt Solution (HBSS) (Gibco) supplemented with fetal calf serum (FCS, 1%, Lonza) and Penicillin/Streptomycin (Gibco) (100 μg/mL) (= wash medium). Epithelial cells were removed by an 8-minute incubation on a magnetic stirrer in warm (37°C) wash medium supplemented with DTT (1 mM) and EDTA (1 mM). After a washing step, tissue was cut in pieces smaller than 3mm and digested by a 25- to 30-min incubation on a magnetic stirrer in warm (37°C) MEM α containing Penicillin/Streptomycin (100 μg/mL), β-mercaptoethanol (50 μM, Sigma-Aldrich®), FCS (5%), DNase (5 U/mL, Roche), Collagenase V (0.85 mg/mL, Sigma-Aldrich®), Collagenase D (1.25 mg/mL, Roche), Dispase (1 mg/mL, Gibco). After digestion, cells were filtered through a 70 μm cell strainer and lamina propria cells were purified using a Percoll (VWR) gradient (44 to 67%). Purified cells were washed and re-suspended in PBS supplemented with FCS (1%) and Penicillin/Streptomycin (100 μg/mL) and stained for Flow Cytometry.
Aguilera-Lizarraga J., Florens M.V., Viola M.F., Jain P., Decraecker L., Appeltans I., Cuende-Estevez M., Fabre N., Van Beek K., Perna E., Balemans D., Stakenborg N., Theofanous S., Bosmans G., Mondelaers S.U., Matteoli G., Martinez S.I., Lopez-Lopez C., Jaramillo-Polanco J., Talavera K., Alpizar Y.A., Feyerabend T.B., Rodewald H.R., Farre R., Redegeld F.A., Si J., Raes J., Breynaert C., Schrijvers R., Bosteels C., Lambrecht B.N., Boyd S.D., Hoh R.A., Cabooter D., Nelis M., Augustijns P., Hendrix S., Strid J., Bisschops R., Reed D.E., Vanner S.J., Denadai-Souza A., Wouters M.M, & Boeckxstaens G.E. (2021). Local immune response to food antigens drives meal-induced abdominal pain. Nature, 590(7844), 151-156.
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10

Isolation and Culture of Trigeminal Ganglia Neurons

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TG neurons were isolated from C57BL/6 mice and cultured as described in previous publication (Liu et al., 2020 (link)) (Malin et al., 2007 (link)). Briefly, TG tissue were dissected and placed in ice-cold Hank’s balanced salt solution (HBSS). Following this, TGs were digested with papain (Worthington, Lakewood, NJ, USA) and collagenase type II/dispase (Thermo Fisher Scientific). TG suspensions were then centrifuged at 1300g for 10 min in Percoll (VWR, Radnor, PA, USA) gradient. After washing twice with complete L15 (L15 with 5% FCS, penicillin/streptomycin, HEPES) and Neurobasal A medium supplemented with 2% B27 supplement (Thermo Fisher Scientific) and 1% penicillin-streptomycin, TG neurons at the bottom were counted and planted on coverslips precoated with poly-d-lysine and laminin. Each coverslip had 3000–4000 neurons. These isolated TG neurons were maintained in Neurobasal A medium supplemented with 2% B27 supplement, 1% penicillin-streptomycin, l-glutamine (500 μM), nerve growth factor (NGF; 50 ng/ml), glial-cell-derived neurotrophic factor (GDNF; 50 ng/ml), and mitotic inhibitor fluorodeoxyuridine (60 μM).
Zhu S., Zidan A., Pang K., Musayeva A., Kang Q, & Yin J. (2022). Promotion of corneal angiogenesis by sensory neuron-derived calcitonin gene-related peptide. Experimental eye research, 220, 109125.
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