Pancoll solution

Manufactured by PAN Biotech

Sourced in Germany

Pancoll solution is a density gradient medium used for the separation and isolation of cells and cell components. It is made up of a mixture of polysaccharides and salts and is designed to create a density gradient that allows the separation of different cell types or subcellular fractions based on their density.

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Lab products found in correlation

Trypsin solution, by Merck Group (2 mentions) Ham s f12 medium, by Merck Group (2 mentions) Ham s f 12, by Merck Group (2 mentions) Pan t cell isolation kit, by Miltenyi Biotec (2 mentions) Anti cd8α alexafluor 647, by BD (1 mentions) Concanavalin a (cona), by Merck Group (1 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions) Igg2a, by BD (1 mentions) Efluor 780, by Thermo Fisher Scientific (1 mentions) Penicillin streptomycin, by PAN Biotech (1 mentions) Dextran t 500, by Carl Roth (1 mentions) Fetal calf serum (fcs), by Merck Group (1 mentions) Recombinant human il 15, by Miltenyi Biotec (1 mentions) Macs gmp t cell transact, by Miltenyi Biotec (1 mentions) Recombinant human il 7, by Miltenyi Biotec (1 mentions) Texmacs gmp medium, by Miltenyi Biotec (1 mentions) Interleukin 7 (il 7), by Miltenyi Biotec (1 mentions) Il 15, by Miltenyi Biotec (1 mentions)

8 protocols using pancoll solution

Porcine PBMC Proliferation Assay

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Mononuclear cells from porcine peripheral blood (PBMC) were isolated by density centrifugation of whole blood diluted 1:2 in 0.9% NaCl using Pancoll solution (density 1.077 g/mL, PAN-Biotech). PBMC were stained with the proliferation marker carboxyfluorescein diacetate succinimidyl ester (CFSE, eBiosciences) at a concentration of 5 mM for 5 min in the dark. CFSE-labeled porcine PBMCs were transferred to IMDM supplemented with 10% FCS and 1% penicillin–streptomycin (all PAN-Biotech, Aidenbach, Germany) and seeded into 96-well round bottom plates (2 Mio cells/200 μL). Proliferation was induced by adding Concanavalin A (ConA, 2 μg/mL; Sigma-Aldrich) and assessed after 5 days by comparing unstimulated and ConA-stimulated PBMC using flow cytometry.
For flow cytometry, cells were stained with the following antibodies specific to pig species: anti-CD4a-Pe-Cy7 (clone 4–12-4, IgG2b, BD Biosciences), anti-CD3ε-PerCP-Cy5.5 (clone BB23-8E6-8C8, IgG2a, BD Biosciences) and anti-CD8α-AlexaFluor^® 647 (clone 76–2-11, IgG2a, BD Biosciences). For dead cell exclusion, a fixable viability dye was used in eFluor^® 780 (eBiosciences). Cells were acquired on BD FACS Canto II with BD FACS Diva software and analyzed using FlowJo v9 software (Tree Star) for proliferative capacity of CD4+ T cells identified as liveCD3⁺CD4⁺CFSE^low.

Rosen K., Ebner F., Schmidt S., Hartmann S., Merle R., Friese A, & Roesler U. (2020). Influence of Immune Status on the Airborne Colonization of Piglets with Methicillin-Resistant Staphylococcus aureus (MRSA) Clonal Complex (CC) 398. European Journal of Microbiology & Immunology, 10(1), 1-10.

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Neutrophil Isolation from Whole Blood

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Human neutrophils were isolated from whole blood by a 3% dextran sedimentation step (Dextran T500, Carl Roth GmbH, Germany) followed by centrifugation over a 1.077 g/ml Pancoll solution (PAN-Biotech). Cell viability was evaluated by trypan blue exclusion, and purity (> 95%) was verified by flow cytometry. After isolation, neutrophils were resuspended in HBSS +/+, quantified, and used for experiments as indicated.

Letsiou E., Teixeira Alves L.G., Fatykhova D., Felten M., Mitchell T.J., Müller-Redetzky H.C., Hocke A.C, & Witzenrath M. (2021). Microvesicles released from pneumolysin-stimulated lung epithelial cells carry mitochondrial cargo and suppress neutrophil oxidative burst. Scientific Reports, 11, 9529.

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Isolation and Purification of Extravillous Trophoblasts

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The protocol for isolating EVTs has been previously described [8 (link)] and adapted from [31 (link)]. After washing first trimester placental samples in Ham’s F12 (Sigma, Hertfordshire, UK), chorionic villi were physically separated and digested in 0.25% (w/v) trypsin solution (Sigma, Hertfordshire, UK). The placental tissue was then filtered and diluted in 25% (v/v) FBS in Ham’s F12 medium (Sigma, Hertfordshire, UK) prior to centrifugation for 5 min at 450× g. Pellets were resuspended and collected prior to being layered over a Pancoll solution (Pan Biotech, Aidenbach, Germany. Density 1.077 g/mL). Samples were spun at 750× g for 20 min to allow for concentration and isolation of the EVT band which was then aspirated and collected into clean tubes prior to a final centrifugation at 500× g. The resulting EVTs were resuspended in trophoblast complete medium (TCM; Ham’s F12 without Phenol red, 20% (v/v) FBS, 100 units and 0.1 mg/mL penicillin/streptomycin, 2 mM L-Glutamine). These cells were then seeded onto 35 mm tissue culture dishes and left to settle overnight before changing the medium to fresh TCM.

Lancaster T., Tabrizi M.E., Repici M., Gupta J, & Gross S.R. (2023). An Extracellular/Membrane-Bound S100P Pool Regulates Motility and Invasion of Human Extravillous Trophoblast Lines and Primary Cells. Biomolecules, 13(8), 1231.

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PBMC Isolation and Cryopreservation

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PBMCs were isolated from each blood sample by density gradient centrifugation. For this purpose, 9 ml of EDTA-whole blood mixed with RPMI 1640 supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin was layered on Pancoll solution (Pan Biotech, Aidenbach, Germany) and centrifuged at 900 x g for 35 minutes with brakes off. Then, the PBMCs (interphase) were transferred to a new 50 ml tube and washed twice with RPMI 1640 medium supplemented with penicillin/streptomycin. Cryostocks with 1x10⁷ PBMCs/ml were prepared in fetal calf serum (FCS) (Sigma Aldrich, St. Louis, MO, USA) supplemented with 10% DMSO.
PBMCs were thawed one day prior to experiments. Up to 90% of viable cells were cultivated in RPMI 1640 with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, and 10 mM HEPES. Cells were incubated at a density of 1x10⁶ cells/ml over night at 37°C, 5% CO₂.

Xie X., Karakoese Z., Ablikim D., Ickler J., Schuhenn J., Zeng X., Feng X., Yang X., Dittmer U., Yang D., Sutter K, & Liu J. (2022). IFNα subtype-specific susceptibility of HBV in the course of chronic infection. Frontiers in Immunology, 13, 1017753.

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Isolation of Extravillous Trophoblasts from First-Trimester Placenta

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The protocol for isolating EVTs was adapted as previously described [35 (link)]. Briefly, first-trimester placental samples were gently washed in Ham’s F12 (Sigma, Welwyn Garden City, UK). Chorionic villi were physically separated and digested in 0.25% (w/v) trypsin solution (Sigma, UK). Placental tissue was filtered and diluted in 25% (v/v) FBS in Ham’s F12 medium (Sigma, UK) before centrifugation for 5 min at 450× g. The supernatant was discarded and the pellets were pooled and resuspended before being layered over a Pancoll solution (Pan Biotech, Aidenbach, Germany. Density 1.077 g/mL). Samples were spun at 750× g for 20 min and the EVT band was aspirated and collected into clean tubes prior to a final centrifugation at 500× g. The resulting EVTs were resuspended in trophoblast complete medium (TCM; Ham’s F12 without Phenol red, 20% (v/v) FBS, 100 units and 0.1 mg/mL penicillin/streptomycin, 2 mM L-Glutamine), seeded onto 35 mm tissue culture dishes, and left to settle overnight before changing the medium to fresh TCM.

Tabrizi M.E., Gupta J.K, & Gross S.R. (2023). Ezrin and Its Phosphorylated Thr567 Form Are Key Regulators of Human Extravillous Trophoblast Motility and Invasion. Cells, 12(5), 711.

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Isolation and Expansion of Human T Cells

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Buffy coats and non-mobilized leukapheresis were obtained from healthy volunteer donors from the DRK Dortmund and Ulm. Human PBMCs were purified by density gradient centrifugation using Pancoll solution (Pan-Biotech, Aidenbach, Germany). Primary human T cells were isolated from PBMCs by negative bead selection according to manufacturer’s recommendations (Pan T cell isolation kit, Miltenyi Biotec, Bergisch Gladbach, Germany) and activated using MACS GMP T Cell TransAct (Miltenyi Biotec, Bergisch Gladbach, Germany). T cells were cultured in TexMACS GMP medium (Miltenyi Biotec, Bergisch Gladbach, Germany) supplemented with 12.5 ng/mL of recombinant human IL-7 and 12.5 ng/mL of recombinant human IL-15 (Miltenyi Biotec, Bergisch Gladbach, Germany). T cells were transduced using lentiviral vectors 24 h after stimulation using an MOI of 5 to maintain the vector copy number of the drug product below 5. T cells were washed 3 days after stimulation to remove MACS GMP T Cell TransAct and lentiviral particles. T cells were expanded for 13–14 days with the addition of fresh culture medium every 2–3 days, before the experiments were started. Timing of T cell generation, including activation, transduction, and addition of medium was kept consistent for experiments comparing multiple donors and CAR constructs.

Kotter B., Engert F., Krueger W., Roy A., Rawashdeh W.A., Cordes N., Drees B., Webster B., Werchau N., Lock D., Dapa S., Schneider D., Ludwig S., Rossig C., Assenmacher M., Mittelstaet J, & Kaiser A.D. (2021). Titratable Pharmacological Regulation of CAR T Cells Using Zinc Finger-Based Transcription Factors. Cancers, 13(19), 4741.

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Isolation and Transduction of Primary Human T Cells

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Primary human T cells were isolated from buffy coats of healthy donors which were obtained from German Red Cross after written consent from volunteers. The local ethics committee of the Medical Faculty Carl Gustav Carus, at the Technische Universität Dresden (Dresden, Germany) approved the research with human T cells (EK138042014). Using density centrifugation with Pancoll solution (1,077 g/ml) (PanBiotech, Aidenbach, Germany), primary T cells were isolated from Human Peripheral Blood Mononuclear Cells (PBMCs) using a pan T cell isolation kit according to the manufacturer's instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). Isolated T cells were stained with fluorescently labeled mAbs against human CD3 (#130-113-138), CD4 (#130-113-225), CD8 (#130-110-683) (Miltenyi Biotec). T cell lentiviral transduction procedure with RevCARs was done as described previously [15] . T cells were cultured with IL-15, IL-7 and IL-2 (Miltenyi Biotec) during transduction, but they were transferred to RPMI complete medium lacking these cytokines one day before any functional assay.

Saleh H.A., Mitwasi N., R Loureiro L., Kegler A., Soto K.E.G., Hoffmann L., Crespo E., Arndt C., Bergmann R., Bachmann M, & Feldmann A. (2024). RevCAR-expressing immune effector cells for targeting of Fn14-positive glioblastoma. Cancer gene therapy.

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Isolation of Peripheral Blood Mononuclear Cells

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Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation.⁴⁴ (link) Briefly, 9 mL whole blood was layered on 5 mL of Pancoll solution (PanBiotech GmbH, Germany) within 4 h of venipuncture and was centrifuged at 800g for 20 min. PBMCs were washed twice with PBS (centrifugation at 300g for 10 min) and a high degree of viability (>90%) was confirmed with trypan blue staining. PBMCs were aliquoted and cryopreserved in a 90% FBS-10% DMSO solution in liquid nitrogen storage.

Balog J.Á., Horti-Oravecz K., Kövesdi D., Bozsik A., Papp J., Butz H., Patócs A., Szebeni G.J, & Grolmusz V.K. (2024). Peripheral immunophenotyping reveals lymphocyte stimulation in healthy women living with hereditary breast and ovarian cancer syndrome. iScience, 27(6), 109882.

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