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Ficoll gradient centrifugation

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Ficoll gradient centrifugation is a laboratory technique used for the separation and purification of specific cell types from complex biological samples, such as blood or tissue. It utilizes a density gradient medium, Ficoll, to facilitate the separation of cells based on their density differences during centrifugation. The core function of this technique is to isolate and concentrate specific cell populations for further analysis or downstream applications.

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7 protocols using ficoll gradient centrifugation

1

Monocyte-Derived Osteoclast Generation

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The study protocol established by Lionetto et al. was used in the experiments [10 (link)]. Ficoll-gradient centrifugation (Amersham Biosciences, Uppsala, Sweden) was used to isolate pooled peripheral blood monocytic cells (PBMCs) from buffy coats of healthy blood donors. RPMI-1640 plus GlutaMAX™ medium (RPMI) (Gibco/Invitrogen, Auckland, New Zealand) supplemented with 5% human serum and 1% antibiotics (10,000 units/mL penicillin G sodium, 10,000 μg/mL streptomycin sulfate, and 25 μg/mL amphotericin B (Gibco standard medium)) was the culture medium for PBMCs at 37°C (humified, 5% CO2, in 25 cm2 tissue culture flasks (Sarstedt)). The nonadherent PBMCs were discarded and the adherent PBMCs were washed twice using 0.1 M phosphate-buffered saline (PBS, pH = 7.2), scraped off, and resuspended in standard medium after 1 h of culturing. These cells were used as monocytic cells (MCs) as described below.
To generate the OC, cell cultures of adherent PBMCs were supplemented with OC differentiation cytokines (10 ng/mL recombinant human macrophage-colony stimulating factor (hM-CSF) and 10 ng/mL recombinant human receptor activator of NF-κB ligand (hRANKL) (ReproTech, Rocky Hill, NJ, USA)).
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2

Isolation and Characterization of Mononuclear Phagocytes

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Approximately 3 mL whole blood were collected in K2-EDTA blood collection tubes (Rich Science, Chengdu, China) from two healthy male volunteers and patients diagnosed with sepsis who provided informed consent according to the protocol approved by the Scientific and Ethics Committee (IMSS reference number XYFY2021-KL248-01). Peripheral blood mononuclear cells (PBMNCs) were isolated from peripheral blood by Ficoll gradient centrifugation (Amersham Biosciences, Freiburg, Germany). Then, the PBMNCs were suspended in RPMI 1640 cell culture media and incubated for 2 h in 5% (v/v) CO2 at 37 °C to allow the attachment of mononuclear phagocytes to the bottom plastic surface as previously described 14 (link), 15 (link). The nonadherent cells were removed. The mononuclear phagocytes were washed with prewarm PBS (Kaiji, Nanjing, China) and collected for the indicated experiments. After some of the sample was fixed in 10% neutral buffered formalin for immunofluorescence, the remaining sample was subjected to mitochondrial protein extraction.
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3

Isolation of Peripheral Blood Mononuclear Cells

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Ficoll gradient centrifugation (Amersham Biosciences, Freiburg, Germany) was used to isolate peripheral blood mononuclear cells (PBMC) from the peripheral blood of study subjects: Peripheral blood was diluted 1:2 with phosphate buffered saline and gently layered on top of the Ficoll solution. Centrifugation was performed at 20 °C with 400 g for 30 min. Then, the cells in the interphase were aspirated and centrifuged at 20 °C with 300 g for 15 min. The supernatant was discarded and the pellet incubated with erythrocyte lysis buffer for 8 min. After that, the cells were washed two times with phosphate buffered saline and centrifuged (20 °C with 300 g for 10 min). Thereafter, PBMC were prepared and analyzed by flow cytometry.
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4

FACS Analysis of PBMC Stem Cells

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Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll gradient centrifugation (Amersham Biosciences, Freiburg, Germany). Cell-surface antigens expression was quantified by Fluorescence Activated Cell Sorting (FACS) analysis as described previously [8 (link)]. The following anti-human monoclonal antibodies have been used: PE-conjugated CD133 (Miltenyi Biotec, Bergisch-Gladbach, Germany), PerCP-conjugated CD34 (BD Biosciences, Heidelberg, Germany), and either FITC-conjugated VCAM-1/CD106, FITC-conjugated ICAM-1/CD54, FITC-conjugated E-selectin/CD62E and FITC-conjugated L-selectin/CD62L. Flow cytometry was executed on a FACSCalibur flow cytometer (BD Biosciences) and data analysis was performed using WinMDI 2·8 software (Scripps Research Institute, La Jolla, CA). CD34+/CD133+-stem cell counts are expressed as percentage of total PBMC in each patient or control.
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5

PBMC Proliferation Assay with CSMSCs

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Peripheral blood mononuclear cells (PBMCs) were isolated by a Ficoll gradient centrifugation (Amersham Biosciences). Mitogen-activated T lymphocyte proliferation was induced by concanavalin A (ConA) or phytohemagglutinin (PHA, all from Sigma-Aldrich) used at a final concentration of 10 μg/mL and 1 μg/mL, respectively, added to 1 × 106 PBMCs. CSMSCs were added to 1 × 106 PBMCs at 104, 2 × 104 and 105 cell numbers and co-cultured for 3 days. On day three, proliferation was detected by a BrDU colorimetric assay directly in the cell culture plate according to the manufacturer’s instructions (Roche, Budapest, Hungary).
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6

Monocyte Isolation and Differentiation

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Peripheral blood mononuclear cells (PBMC) were separated from buffy coats by Ficoll gradient centrifugation (Amersham Biosciences, Uppsala, Sweden). Monocytes were isolated by positive selection using magnetic cell separation with anti-CD14-conjugated microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany). The proportion of CD14+ cells was determined by flow cytometry and was found to be 96–99% dependent on the blood donor. The purified cells were cultured at a density of 106 cells/mL in serum free AIMV medium supplemented with 80 ng/mL granulocyte-macrophage colony stimulating factor (GM-CSF) purchased from Gentaur and 50 ng/mL IL-4 from Peprotech.
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7

Measuring MSC Immunomodulatory Capacity

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Peripheral blood mononuclear cells (PBMCs) were isolated by a Ficoll gradient centrifugation (Amersham Biosciences). Prior to the test, 104 and 105 MSCs were placed in the cell culture plates, and nonadherent cells were removed by a gentle wash step. PBMCs required for the MLR test (1 × 106) were added 24 hours later. Mitogen-activated T lymphocyte proliferation was induced by addition of concanavalin A (ConA) or phytohemagglutinin (PHA, both from Sigma-Aldrich) at a final concentration of 10 μg/ml and 1 μg/ml, respectively, to the MSC-PBMC cocultures. On day three, proliferation was detected by a BrDU colorimetric assay directly in the cell culture plate according to the manufacturer's instructions (Roche, Budapest, Hungary). In control experiments, MSCs and PBMCs were cultured together or separately with and without mitogenic activation. To compare the immunosuppressive capacity of SV-MSCs and BM-MSCs, the proliferation of mitogen-activated PBMCs (OD values, BrdU incorporation) was taken as value 1, and changes in BrdU incorporation caused by MSCs were compared.
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