Percoll gradient

Manufactured by GE Healthcare

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Percoll gradient is a colloidal silica-based medium used for the separation and isolation of cells, organelles, and other biological particles through density gradient centrifugation. It provides a gentle, non-toxic environment that preserves the integrity and function of the separated components.

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Ficoll-Percoll gradients (2 citations) Percoll PLUS gradient (3 citations) Percoll gradient separation (8 citations) Two-step Percoll gradient (2 citations) Percoll density gradient centrifugation (30 citations) Percoll gradient medium (4 citations) Discontinuous Percoll gradient (12 citations) Percoll density gradient (39 citations) Percoll gradient centrifugation (37 citations) Percoll density gradient centrifugation media (3 citations)

290 protocols using percoll gradient

Intestinal Cell Isolation Protocol

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The intestine was dissected and surrounding fat pads and peyer’s patches were surgically removed. Small and large intestine were separated and both pieces were opened longitudinally and washed in RPMI containing 10% FBS and HEPES (2X, Thermo Scientific). The intestinal compartments were cut into small pieces and collected in 30ml RPMI with 10% FBS and HEPES (2X, Thermo Scientific). Subsequently, EDTA (Milipore Sigma) and DTT (Milipore Sigma) were added to a final concentration of 5mM and 1mM, respectively. The suspension was incubated for 25min at 37°C while shaking at 250rpm and subsequently filtered through a 70μm strainer. After washing in PBS, the intestinal cell pellet was resuspended in 4ml of a 40% percoll gradient (GE Healthcare). The 40% percoll suspension was layered on top of 5 ml of a 60% percoll gradient (GE Healthcare) and centrifuged at room temperature at 900xg for 25min without acceleration and brake. The cell layer at the interphase was collected, washed with PBS, and kept on ice for staining with fluorochrome-conjugated antibodies and flow cytometric analysis.

Winkels H., Ghosheh Y., Kobiyama K., Kiosses W.B., Orecchioni M., Ehinger E., Suryawanshi V., Herrera-De La Mata S., Marchovecchio P., Riffelmacher T., Thiault N., Kronenberg M., Wolf D., Seumois G., Vijayanand P, & Ley K. (2021). Thymus-derived CD4+CD8+ cells reside in mediastinal adipose tissue and the aortic arch. Journal of immunology (Baltimore, Md. : 1950), 207(11), 2720-2732.

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Isolation and Purification of Cryptosporidium Sporozoites

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The release of sporocysts was achieved by vortexing sporulated oocysts (2.5 × 10⁷/mL) at 2000 rpm with 1 mm-diameter glass beads (Sigma, St. Louis, MO, USA) for 70 s. Sporocysts were purified using a 50% Percoll gradient (density: 1.13 g/mL, GE Healthcare, Piscataway, NJ, USA), resuspended at 1 × 10⁶/mL in excystation medium, and incubated at 42 °C for 150 min. Freshly excysted sporozoites were washed and purified on a 60% Percoll gradient (density: 1.13 g/mL, GE Healthcare, Piscataway, NJ, USA) [23 (link)]. Sporozoites for the immunization schedule were resuspended in sterile PBS, gradually frozen at −70 °C at a rate of 1 °C/min, and stored at −70 °C until use.

Juárez-Estrada M.A., Gayosso-Vázquez A., Tellez-Isaias G, & Alonso-Morales R.A. (2021). Protective Immunity Induced by an Eimeria tenella Whole Sporozoite Vaccine Elicits Specific B-Cell Antigens. Animals : an Open Access Journal from MDPI, 11(5), 1344.

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Isolation of Intestinal Epithelial and Lamina Propria Lymphocytes

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Small intestines were dissected from mice, flushed with the ice-cold PBS, and cut open longitudinally. The intestines were cut into small sections that were thoroughly washed with ice-cold PBS. Tissues were incubated at 37°C in Hank’s buffered salt solution (HBSS) supplemented with 3% FBS, 1 mM EDTA, and 1 mM DTT. After 30 min, tissues were vortexed for 2 min, and the cell suspensions containing intestinal epithelial cells and the IELs were filtered through a 100-μm cell strainer followed by passage through a glass wool column (Ohio Valley). The cell suspensions were washed and suspended in ice-cold RPMI 1640, and then applied to a 40%:80% Percoll gradient (GE Healthcare). After centrifugation at 800g for 20 min, the IELs were collected from the Percoll gradient interface and prepared for RNA isolation and purification.
For LPL isolation, the residual tissue fragments were digested for 1 hour at 37°C in RPMI 1640 containing 0.025 mg/ml Collagenase IV (Sigma-Aldrich), 0.05 mg/ml DNase I (Sigma-Aldrich), and 0.25 units/ml Dispase (BD Biosciences). Cells were filtered through 40-μm cell strainers and applied to a 40%:80% Percoll gradient (GE Healthcare). After centrifugation, the LPL were collected from the gradient interface and prepared for RNA isolation and purification.

Hu Z., Zhang C., Sifuentes-Dominguez L., Zarek C.M., Propheter D.C., Kuang Z., Wang Y., Pendse M., Ruhn K.A., Hassell B., Behrendt C.L., Zhang B., Raj P., Harris-Tryon T.A., Reese T.A, & Hooper L.V. (2021). Small proline-rich protein 2A is a gut bactericidal protein deployed during helminth infection. Science (New York, N.Y.), 374(6568), eabe6723.

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Isolation of PBMCs from Buffy Coats

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The isolation of PBMCs from buffy coats was adapted from Repnik U et al.80 (link). Briefly, buffy coats were obtained from the Central Institute for Transfusion Medicine, Asklepios Klinik Hamburg (Germany) and WBCs were purified using a Ficoll gradient (GE healthcare, Uppsala, Sweden), a subsequent hyperosmotic Percoll gradient (GE healthcare, Uppsala, Sweden) led to separation of monocytes from lymphocytes and a third iso-osmotic Percoll gradient to monocytes from platelets and dead cells. The pellet obtained after this gradient is the monocyte-enriched fraction, which we refer to as PBMCs. According to the forward and side scatter plots, this fraction contains about 55–80% monocytes along with 20–45% of lymphocytes.

Dharmadhikari G., Stolz K., Hauke M., Morgan N.G., Varki A., de Koning E., Kelm S, & Maedler K. (2017). Siglec-7 restores β-cell function and survival and reduces inflammation in pancreatic islets from patients with diabetes. Scientific Reports, 7, 45319.

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Sperm Purification and Quantification

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After liquefaction, each semen sample was analyzed according to the World Health Organization criteria (WHO, 2010). Patients were excluded upon identification of any significant androgenic or endocrine abnormalities. The patients found to have chromosomal aberrations by karyotype testing were also excluded. Samples with abnormal pH and viscosity, increased number of leukocytes or immature germ cells, and abnormal semen liquefaction were excluded. Asthenozoospermia was defined as the progressive motility of sperm < 32 % within 60 min of ejaculation. The fresh human semen specimens were centrifuged using a two-layer gradient system including an upper layer solution of 40 % Percoll gradient and a lower layer solution of 80 % Percoll gradient (GE Healthcare, Waukesha, WI, USA) (400 g, 15 min) to separate spermatozoa from seminal plasma. Spermatozoa were then washed twice with 2.0 ml PBS at 300 g for 5 min. Each sample was diluted to 1.0 ml with PBS, and then the sperms count were calculated by a hemocytometer as previously described [30 ]. Subsequently, 100 µL of sperm solution was prepared at a concentration of 1 × 10⁸ cells/ml for lipid extraction.

Chen S., Wang M., Li L., Wang J., Ma X., Zhang H., Cai Y., Kang B., Huang J, & Li B. (2021). High-coverage targeted lipidomics revealed dramatic lipid compositional changes in asthenozoospermic spermatozoa and inverse correlation of ganglioside GM3 with sperm motility. Reproductive Biology and Endocrinology : RB&E, 19, 105.

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Single-cell Sequencing Preparation from Mouse Brain

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To prepare cells for single-cell sequencing, adult mice (8–20 weeks) were anesthetized with pentobarbital and subsequently perfused with 5 units/mL heparin in saline for approximately 1 min. Brains were rapidly dissected and finely minced. For single-cell sequencing experiments, tissue was digested in HBSS with calcium and magnesium (Gibco, 14025-092) supplemented with 20 units per mL papain (Worthington Biochemical LS003126) and 50 units per mL DNase (Worthington Biochemical, LS002139). Tissue was digested at 37 °C with gentle shaking for 45 min, with trituration after every 15-min interval to dissociate the tissue. Following digestion, a 40% Percoll gradient (GE Healthcare, 17-0891-01) was used to remove myelin and other debris from the samples. Resulting single-cell suspensions from 4 to 5 mice were pooled for each sequencing sample and subsequently stained for FACS sorting.

Beiter R.M., Rivet-Noor C., Merchak A.R., Bai R., Johanson D.M., Slogar E., Sol-Church K., Overall C.C, & Gaultier A. (2022). Evidence for oligodendrocyte progenitor cell heterogeneity in the adult mouse brain. Scientific Reports, 12, 12921.

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Isolation of Liver Mononuclear Cells

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Liver MNCs were isolated as described previously [12 (link),23 (link)]. Briefly, liver tissues were dissociated by mashing through a 70 μm nylon mesh cell strainer (BD Falcon, Millville, NJ). The liver cell suspension was suspended in phosphate-buffered saline (PBS) and centrifuged at 42 g for 5 minutes to eliminate hepatocytes. The supernatant was collected, washed in PBS, and resuspended in 40% Percoll gradient (GE Healthcare, Buckinghamshire, United Kingdom) in PBS. The cell suspension was centrifuged at 1,200 g for 30 minutes at 4°C. The supernatant was removed by mechanical suction, and MNCs were collected, washed in PBS, and resuspended in RPMI-1640 medium. For the isolation of hepatic NK cells, CD3^- liver MNCs were separated from total liver MNCs by negative magnetic cell sorting (Miltenyi Biotec, Auburn, CA). NK1.1⁺CD3^- cells were then purified with anti-NK1.1 monoclonal antibodies by positive magnetic cell sorting. Approximately 95% of cells purified this way were NK1.1⁺CD3^- cells.

Yi H.S., Eun H.S., Lee Y.S., Jung J.Y., Park S.H., Park K.G., Choi H.S., Suh J.M, & Jeong W.I. (2015). Treatment with 4-Methylpyrazole Modulated Stellate Cells and Natural Killer Cells and Ameliorated Liver Fibrosis in Mice. PLoS ONE, 10(5), e0127946.

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Isolation of Tumor-Infiltrating Lymphocytes

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B16-F10 melanoma cells (2.5 × 10⁵ cells/100 μl of PBS) were subcutaneously injected into the side flank of the C57/BL6 mice (Day 0). Seven days after tumor cell injection, we started to measure the size of the tumors (length and wide) and intraperitoneally injected DMSO-PBS, curcumin-PBS (5 mg/kg), or GO-Y030-PBS (5 mg/kg). Tumor volume (mm³) was calculated using the following formula: 0.5 × length (mm) × width (mm) × width (mm). Thirteen to sixteen days after tumor cell injection, we harvested the tumors and washed them with pre-cooled PBS. Then, the tumors were cut using scissors and digested using 1.25 mg/ml collagenase IV (Roche, Rotkreuz, Switzerland) and 0.1 mg/ml DNase I (Sigma-Aldrich) in PBS at 37°C in a shaker for 1 h. After digestion, we added 15 ml of PBS to the tumors and washed the digested tumors by centrifugation (300 × g, 5 min, 4°C). To purify the tumor-infiltrating lymphocytes, we re-suspended the tumor digestion using a 40% Percoll gradient (GE Healthcare, Chicago, IL) and added an 80% Percoll gradient into the bottom, followed by centrifugation at 550 × g for 30 min at 4°C. Then, we collected the middle layers and immediately washed them with PBS.

MaruYama T., Kobayashi S., Nakatsukasa H., Moritoki Y., Taguchi D., Sunagawa Y., Morimoto T., Asao A., Jin W., Owada Y., Ishii N., Iwabuchi Y., Yoshimura A., Chen W, & Shibata H. (2021). The Curcumin Analog GO-Y030 Controls the Generation and Stability of Regulatory T Cells. Frontiers in Immunology, 12, 687669.

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Isolation and Culture of Cytotrophoblasts from Human Placenta

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CTBs were isolated from human term placentas as previously described [23 ]. Briefly, the placental tissues were enzymatically digested through incubation with dispase (Worthington, Lakewood, NJ, USA), 0.25 % trypsin (Gibco), and 0.02 % DNase (Sigma, Missouri, USA) in a 37 °C water bath for 45 min in a way to remove the outer syncytium layer and release the underlying CTBs. Cells were purified via centrifugation at 1400 rpm for 30 min on a 40 % Percoll gradient (GE Healthcare Biosciences, USA) that had been diluted in 10 % 10×HBSS buffer (diluted in ddH₂O) and pre-centrifuged at 15,000 rpm for 50 min. The cytotrophoblast layer was just above the red blood cell layer. The isolated CTBs were incubated at 37 °C, 5 % CO₂ for 3 h to allow for cell adhesion or for 48 h to allow for cell syncytialization.

Peng W., Liu Y., Qi H, & Li Q. (2021). Alpha‐actinin‐4 is essential for maintaining normal trophoblast proliferation and differentiation during early pregnancy. Reproductive Biology and Endocrinology : RB&E, 19, 48.

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Isolation and Purification of Hepatic Leukocytes and Hepatocytes

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Hepatic leukocytes were isolated as described previously [24 (link)]. Briefly, liver tissues were minced and sieved through a 70 μm filter. Hepatic leukocytes were purified by centrifugation on a 40% Percoll gradient (GE Healthcare), and red blood cells were lysed by RBC lysis buffer. Collected leukocytes were further enriched using PE-anti-F4/80 (BD Pharmingen, Cat# 565410) and anti-PE microbeads (Miltenyi Biotec) to isolate macrophages. Hepatocytes were isolated as described previously [25 (link)]. Briefly, the liver was perfused with a solution of EGTA and digested with a 0.075% collagenase solution. The viable hepatocytes were separated by 40% Percoll solution with centrifugation at 420 × g for 10 min at 4 °C.

Hou X., Liu Q., Gao Y., Yong L., Xie H., Li W., Zhou Y., Liu J., Feng L., Xu L., Shen Y, & Wang H. (2022). Mesencephalic astrocyte-derived neurotrophic factor reprograms macrophages to ameliorate acetaminophen-induced acute liver injury via p38 MAPK pathway. Cell Death & Disease, 13(2), 100.

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