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Ficoll paque cushion

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Ficoll-Paque cushion is a sterile, pyrogen-free medium used for the separation and isolation of mononuclear cells from whole blood or other cell suspensions by density gradient centrifugation.

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Lab products found in correlation

Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (2 mentions) Isotype control antibody, by BD (1 mentions) Anti human cd3, by BD (1 mentions) Anti human cd4, by BD (1 mentions) Anti human cd8, by BD (1 mentions) Annexin 5, by BD (1 mentions) Facsverse flow cytometer, by BD (1 mentions) Rpmi 1640, by Thermo Fisher Scientific (1 mentions)

6 protocols using ficoll paque cushion

1

Immunophenotyping of Blood and PBMCs

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Plasma was separated from EDTA-contained fresh blood samples, aliquoted, and stored at − 80 °C. Peripheral blood mononuclear cells (PBMCs) were isolated over a Ficoll-Paque cushion (GE Healthcare, Wauwatosa, WI). PBMCs were used for annexin V assays. Blood samples were used for all other flow cytometry-based assays except annexin V assays. For surface staining, antibodies were incubated with blood or PBMCs at room temperature for 15 min. After surface staining in blood samples, red cells were lysed, washed, and analyzed by flow cytometry. The fluorochrome-labeled mAbs (BD Pharmingen, San Jose, CA) used for flow cytometry included the following: anti-human CD3 (OKT3), anti-human CD4 (RPA-T4), anti-human CD8 (RPA-T8), anti-human CD19 (HIB19), anti-human CD20 (L27), anti-human CD27 (M-T271), anti-human CD38 (HIT2), anti-human CD45RA (HI100), anti-human HLA-DR (G46-6), anti-human ki67 (B56), anti-human IgD (IA6-2), anti-human IgG (G18-145), isotype control antibodies (BD Pharmingen), and annexin V (BD Pharmingen). Cells were collected in a BD FACSVerse flow cytometer (BD, San Jose, CA), and data was analyzed by FlowJo software (Version 10.0.8).
Luo Z., Li M., Wu Y., Meng Z., Martin L., Zhang L., Ogunrinde E., Zhou Z., Qin S., Wan Z., Westerink M.A., Warth S., Liu H., Jin P., Stroncek D., Li Q.Z., Wang E., Wu X., Heath S.L., Li Z., Alekseyenko A.V, & Jiang W. (2019). Systemic translocation of Staphylococcus drives autoantibody production in HIV disease. Microbiome, 7, 25.
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2

Monocyte-derived Macrophage Differentiation

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Blood was collected from healthy human volunteers under a protocol approved by the Ohio State University’s Office of Responsible Research Practices (IRB), with written donor consent. Human peripheral blood mononuclear cells (PBMCs) were then isolated from heparinized blood on a Ficoll-Paque cushion (GE Healthcare) as previously described [25 (link)]. Differentiation of monocytes into monocyte-derived macrophages (MDMs) was accomplished by incubation of PBMCs (2x106/mL) within Teflon wells (Savillex) at 37°C with 5% CO2 over a period of five days in RPMI 1640 medium containing 20% autologous serum, which contains < 3 μM zinc. MDMs in the PBMCs were placed in monolayer culture (99% pure), washed and repleted with RPMI containing 2% autologous serum or 10% autologous serum, then repleted with or without 10 μM, 18 μM or 40 μM Zinc Sulfate, with or without LPS 100 ng/mL.
Pyle C.J., Akhter S., Bao S., Dodd C.E., Schlesinger L.S, & Knoell D.L. (2017). Zinc Modulates Endotoxin-Induced Human Macrophage Inflammation through ZIP8 Induction and C/EBPβ Inhibition. PLoS ONE, 12(1), e0169531.
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3

Preparation and Cultivation of Monocyte-Derived Macrophages

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MDMs were prepared as described elsewhere [69 (link),70 (link)]. Briefly, heparinized blood was layered on a Ficoll-Paque cushion (GE Healthcare, Uppsala, Sweden) to allow for collection of PBMCs. PBMCs were cultured in RPMI (Life Technologies, Carlsbad, CA) with 20% autologous serum in Teflon wells (Savillex, Eden Prairie, MN) for 5 days at 37°C/5% CO2. MDMs were harvested and adhered to tissue culture dishes for 2–3 h in RPMI with 10% autologous serum, lymphocytes were washed away, and MDMs were incubated overnight in RPMI with 10% autologous serum. Such MDM monolayers are 99% pure and viable.
Arnett E., Weaver A.M., Woodyard K.C., Montoya M.J., Li M., Hoang K.V., Hayhurst A., Azad A.K, & Schlesinger L.S. (2018). PPARγ is critical for Mycobacterium tuberculosis induction of Mcl-1 and limitation of human macrophage apoptosis. PLoS Pathogens, 14(6), e1007100.
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4

Macrophage-mediated EBOV infection assay

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Monocyte derived macrophages were isolated as described previously (30 (link), 31 (link)).Briefly, human peripheral blood was collected from healthy donors according to University of Texas Health approved IRB protocol #20180013HU. Heparinized blood was layered on a Ficoll-Paque cushion (GE Healthcare, Uppsala, Sweden) to allow for collection of peripheral blood mononuclear cells (PBMCs). PBMCs were cultured in Teflon wells (Savillex, Eden Prairie, MN) RPMI (Life Technologies, Carlsbad, CA) with 20% autologous serum for 5 days at 37°C/5% CO2. PBMCs were pelleted, resuspended in RPMI with 10% autologous serum, and plated into wells of a 96-well plate overnight. Subsequently, cells were treated as described in the figure legends for 24 hours. Stimulated MDMs were incubated with EBOV at MOI=0.1 for 30 min to allow binding, then washed, and overlaid with fresh medium. The cells were fixed after 24 hours. The supernatants were then titrated on Vero cell monolayers for 24 hours. Subsequently, all samples were treated with anti-VP40 antibody to identify infected cells and Hoescht dye to stain nuclei. Imaging and analysis were performed as described above.
Rogers K.J., Shtanko O., Stunz L.L., Mallinger L.N., Arkee T., Schmidt M.E., Bohan D., Brunton B., White J.M., Varga S.M., Butler N.S., Bishop G.A, & Maury W. (2020). CD40 signaling restricts RNA virus replication in macrophages, leading to rapid innate immune control of acute virus infection. Journal of leukocyte biology, 109(2), 309-325.
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5

Monocyte-Derived Macrophage Isolation Protocol

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MDMs were prepared as described elsewhere [25 (link),26 (link)]. Briefly, heparinized blood was layered on a Ficoll-Paque cushion (GE Healthcare, Uppsala, Sweden) to allow for collection of PBMCs. PBMCs were cultured in RPMI 1640 (Life Technologies, Carlsbad, CA) with 20% autologous serum in Teflon wells (Savillex, Eden Prairie, MN) for 5 days at 37 ◦C/5% CO2. MDMs were harvested and adhered to tissue culture dishes for 2–3 h in RPMI with 10% autologous serum, lymphocytes were washed away, and MDMs were incubated overnight in RPMI with 10% autologous serum. Such MDM monolayers are 99% pure and viable.
Arnett E., Pahari S., Wager C.M., Hernandez E., Bonifacio J.R., Lumbreras M., Renshaw C., Montoya M.J., Opferman J.T, & Schlesinger L.S. (2023). Combination of MCL-1 and BCL-2 inhibitors is a promising approach for a host-directed therapy for tuberculosis. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie, 168, 115738.
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6

Monocyte-Derived Macrophage Isolation

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MDMs were prepared as described elsewhere [74 ]. Briefly, heparinized blood was layered on a Ficoll-Paque cushion (GE Healthcare, Uppsala, Sweden) to allow for collection of PBMCs. PBMCs were cultured in RPMI (Life Technologies, Carlsbad, CA) with 20% autologous serum in Teflon wells (Savillex, Eden Prairie, MN) for 5 days at 37°C/5% CO2. MDMs were harvested and adhered to tissue culture dishes for 2–3 h in RPMI with 10% autologous serum, lymphocytes were washed away, and MDMs were incubated overnight in RPMI with 10% autologous serum. Such MDM monolayers are 99% pure and viable.
Leopold Wager C.M., Bonifacio J.R., Simper J., Naoun A.A., Arnett E, & Schlesinger L.S. (2023). Activation of transcription factor CREB in human macrophages by Mycobacterium tuberculosis promotes bacterial survival, reduces NF-kB nuclear transit and limits phagolysosome fusion by reduced necroptotic signaling. PLOS Pathogens, 19(3), e1011297.
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