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Ficoll paque plus separation

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Ficoll-Paque Plus is a density gradient medium used for the separation and isolation of cells, organelles, and other biological particles. It is a sterile, pyrogen-free, and endotoxin-tested solution that utilizes the principles of density gradient centrifugation to effectively separate different cell types based on their density. The product provides a simple and efficient method for the isolation of various cell populations, such as mononuclear cells, from whole blood or other biological samples.

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Lab products found in correlation

Pbs edta, by Thermo Fisher Scientific (2 mentions) Countess 2 automated cell counter, by Thermo Fisher Scientific (1 mentions) Penicillin, by Thermo Fisher Scientific (1 mentions) Streptomycin, by Thermo Fisher Scientific (1 mentions) Bryostatin 1, by Bio-Techne (1 mentions) Cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions) Fetal bovine serum (fbs), by Omega Scientific (1 mentions) Prostratin, by LC Laboratories (1 mentions) Magnetic activated cell sorting (macs), by Miltenyi Biotec (1 mentions) Cd3 pacific blue sp34 2, by BD (1 mentions) Cd14 pe hcd14, by BioLegend (1 mentions) Pkh67 green fluorescent cell linker kit, by Merck Group (1 mentions) Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions) Lsrfortessa, by BD (1 mentions) Facsaria, by BD (1 mentions)

Close spelling variants of this product

Ficoll-Paque Plus separation medium Ficoll-paque PLUS gradient separation Ficoll-Paque separation Ficoll-Paque PLUS Ficoll-Paque Ficoll separation Ficoll

6 protocols using ficoll paque plus separation

1

Isolation of Peripheral Blood Mononuclear Cells

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Peripheral whole blood was collected in EDTA tubes and processed fresh using Ficoll‒Paque Plus separation (GE Healthcare, Barrington, IL, USA, 17144002). The blood was first diluted with 5 mL of 2 mM EDTA‒PBS (Invitrogen, Carlsbad, CA, USA, 1555785-038) before 10–20 mL of diluted blood was carefully layered onto 15 mL of Ficoll in a 50 mL Falcon tube. The samples were centrifuged at 900 g for 30 min at room temperature (22 °C–24 °C). The plasma layer was carefully separated, and the PBMC layer was collected using a sterile Pasteur pipette. The PBMC layer was washed with three volumes of EDTA-PBS by centrifugation at 500 g for 5 min. The pellet was resuspended in EDTA-PBS and centrifuged at 400 g for 5 min. The PBMC pellet was collected, and the cell number and viability were assessed using Trypan blue and a Countess II Automated Cell Counter (Thermo Fisher Scientific, Waltham, MA, USA).
Sung M., Kim J.H., Min H.S., Jang S., Hong J., Choi B.K., Shin J., Chung K.S, & Park Y.R. (2023). Three-dimensional label-free morphology of CD8 + T cells as a sepsis biomarker. Light, Science & Applications, 12, 265.
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2

Activation of Primary CD4+ T Cells

Cited 1 time
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Primary human cells from HIV seronegative individuals were cultured in “RF10 medium” consisting of RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS, Omega Scientific), 100 U/mL of penicillin, and 100 μg/mL of streptomycin (Invitrogen). PBMCs were isolated using Ficoll-Paque Plus separation (GE Healthcare). Primary CD4+ T cells were separated from PBMCs by negative immunomagnetic selection using the CD4+ T cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instructions. Cells were then exposed to compound for 24 hours before staining and flow cytometric analysis of CD69 levels. Compound incubations were performed by seeding 105 cells/well in 100 μL of RF10 media containing the appropriate concentration of compound in wells of a v-bottomed 96-well plate. Bryostatin 1 (Tocris bioscience) and Prostratin (LC Laboratories) were obtained commercially, and bryologs or Prostratin analogs were synthesized as previously described [18 (link),26 (link)].
Marsden M.D., Loy B.A., Wu X., Ramirez C.M., Schrier A.J., Murray D., Shimizu A., Ryckbosch S.M., Near K.E., Chun T.W., Wender P.A, & Zack J.A. (2017). In vivo activation of latent HIV with a synthetic bryostatin analog effects both latent cell "kick" and "kill" in strategy for virus eradication. PLoS Pathogens, 13(9), e1006575.
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3

Isolation of B Cells from PBMCs

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Peripheral blood in EDTA-tubes (15–20 ml) was obtained before (range 6 to 12 weeks) and after (range 13 to 16 months) aHSCT and processed with Ficoll-Paque Plus separation (GE Healthcare, Munich, Germany) according to the manufacturer’s instructions to receive PBMCs. PBMCs were stored in liquid nitrogen before they were incubated with CD19 monoclonal antibody-coupled microbeads to separate B cells by magnetic cell sorting (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany). Each PBMC sample was loaded onto two MACS columns successively to achieve a B cell purity over 95%.
Gernert M., Tony H.P., Schwaneck E.C., Gadeholt O, & Schmalzing M. (2019). Autologous hematopoietic stem cell transplantation in systemic sclerosis induces long-lasting changes in B cell homeostasis toward an anti-inflammatory B cell cytokine pattern. Arthritis Research & Therapy, 21, 106.
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4

Exosome Binding Assay with PBMCs

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PBMCs were isolated from buffy coat preparations of healthy blood donors (Blood Transfusion Center Solna, Stockholm, Sweden) through Ficoll-Paque Plus separation (GE Healthcare), as previously described (25 (link)). Exosomes (10 µg) were stained with a PKH67 Green Fluorescent Cell Linker Kit (Sigma-Aldrich), as previously described (25 (link)). Prefiltered (0.22-µm filter) PKH67-stained exosomes were added to PBMCs (2.5 × 105) for 1, 2, or 4 h at 37°C, 5% CO2. A PKH67 dye pellet centrifuged in parallel with labeled exosomes served as negative background control. PBMCs were stained with the following Abs to distinguish B cells (CD3−CD19+HLA-DR+), monocytes (CD3−CD14+HLA-DR+), and T cells (CD3+CD19−): CD19-ECD (HD237; B4 lytic; Beckman Coulter); HLA-DR–PE-Cy5 (TU36; BD Biosciences), CD14-PE (HCD14; BioLegend), and CD3 Pacific Blue (SP34-2; BD Biosciences). Exosome binding to live PBMCs (LIVE/DEAD Fixable Aqua Dead Cell Stain Kit; Invitrogen) was measured (~150,000 events) using an LSR Fortessa (BD) or FACSAria (BD) and analyzed using FlowJo software.
Gutzeit C., Nagy N., Gentile M., Lyberg K., Gumz J., Vallhov H., Puga I., Klein E., Gabrielsson S., Cerutti A, & Scheynius A. (2014). Exosomes Derived from Burkitt’s Lymphoma Cell Lines Induce Proliferation, Differentiation, and Class-Switch Recombination in B Cells. Journal of immunology (Baltimore, Md. : 1950), 192(12), 5852-5862.
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5

Isolation and Purification of B Cells

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Ficoll-Paque Plus separation (GE Healthcare, Munich, Germany) was used according to the manufacturer’s instructions to isolate PBMCs out of 15–20 ml peripheral blood from EDTA-tubes. PBMCs were stored in liquid nitrogen before further processing. Magnetic cell sorting (MACS) with CD19 monoclonal antibody-coupled microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) was performed. PBMCs were twice positively selected (i.e., two consecutive columns) for CD19 to achieve a purity of more than 87% of B cells for the B cell cultures.
Gernert M., Tony H.P., Schwaneck E.C., Gadeholt O., Fröhlich M., Portegys J., Strunz P.P, & Schmalzing M. (2021). Lymphocyte subsets in the peripheral blood are disturbed in systemic sclerosis patients and can be changed by immunosuppressive medication. Rheumatology International, 42(8), 1373-1381.
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6

PBMC Isolation from Whole Blood

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Peripheral whole blood was collected in EDTA tubes and processed fresh via Ficoll-Paque Plus separation (GE Healthcare, 17144002). The blood was first diluted with 5 ml 2 mM EDTA-PBS (Invitrogen, 1555785-038), before 10–20 ml of diluted blood was carefully layered onto 15 ml of Ficoll in a 50-ml falcon tube. If the sample volume was less than 5 ml, blood was diluted with an equal volume of EDTA-PBS and layered onto 3 ml Ficoll. The sample was centrifuged at 800g for 20 min at room temperature. The plasma layer was carefully removed and the PBMC layer collected using a sterile Pasteur pipette. The PBMC layer was washed with three volumes of EDTA-PBS by centrifugation at 500g for 10 min. The pellet was suspended in EDTA-PBS and centrifuged again at 300g for 5 min. The PBMC pellet was collected and the cell number and viability assessed using Trypan blue. Cell freezing medium (90% FBS and 10% DMSO) was added dropwise to PBMCs slowly on ice and the mixture cryopreserved at −80 °C until further full-sample processing.
Stephenson E., Reynolds G., Botting R.A., Calero-Nieto F.J., Morgan M.D., Tuong Z.K., Bach K., Sungnak W., Worlock K.B., Yoshida M., Kumasaka N., Kania K., Engelbert J., Olabi B., Spegarova J.S., Wilson N.K., Mende N., Jardine L., Gardner L.C., Goh I., Horsfall D., McGrath J., Webb S., Mather M.W., Lindeboom R.G., Dann E., Huang N., Polanski K., Prigmore E., Gothe F., Scott J., Payne R.P., Baker K.F., Hanrath A.T., Schim van der Loeff I.C., Barr A.S., Sanchez-Gonzalez A., Bergamaschi L., Mescia F., Barnes J.L., Kilich E., de Wilton A., Saigal A., Saleh A., Janes S.M., Smith C.M., Gopee N., Wilson C., Coupland P., Coxhead J.M., Kiselev V.Y., van Dongen S., Bacardit J., King H.W., Rostron A.J., Simpson A.J., Hambleton S., Laurenti E., Lyons P.A., Meyer K.B., Nikolić M.Z., Duncan C.J., Smith K.G., Teichmann S.A., Clatworthy M.R., Marioni J.C., Göttgens B, & Haniffa M. (2021). Single-cell multi-omics analysis of the immune response in COVID-19. Nature Medicine, 27(5), 904-916.
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